I. INTRODUCTION

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Mammalian kidneys play a major role in the homeostasis of extracellular compartment. Despite large qualitative and quantitative variations in dietary intake of solutes and water, the kidneys are able to maintain the composition and the volume of the extracellular compartment within very narrow margins. This homeostatic function of kidneys requires the presence of numbers of specific carriers able to transport a large variety of substrates and their fine control by specific factors and hormones.

Since the onset of modern renal physiology, tremendous efforts have been made to describe the transport properties of the kidney tubule and to analyze their regulatory factors. This led, in the mid 1980s, to an almost coherent cellular description of the transport properties of the successive segments constituting the nephron, as well as to the localization and characterization of hormonal regulation of these processes (566).

During the past 10-15 years, most efforts have permitted the evolution from this cellular level of understanding to a molecular one. This evolution mainly results from 1) the molecular cloning of membrane transporters and hormone receptors involved in solute and water transport and its regulation, 2) the characterization of new extracellular regulatory factors and deciphering of new intracellular signaling pathways, 3) the acknowledgement that intracellular signaling pathways should not be considered as linear and parallel chains of interactions, but as intricated and interactive networks. Such combinatorial organization markedly increases the diversity of signaling. 4) Lastly, the idea has slowly emerged that the cornerstone of kidney transport machinery, Na⁺-K⁺-ATPase, is not a house-keeping protein that does not participate actively to rapid adaptations of kidney function but is an important molecular target of hormonal regulation. For this last reason, we have chosen to take Na⁺-K⁺-ATPase as a leading thread in the analysis of the hormonal control of sodium transport in the kidney.

This review has been focused on the regulatory pathways of sodium transport that share the following criteria: 1) mechanisms are deciphered, at least partially, at the molecular level; 2) transport is dependent on Na⁺-K⁺-ATPase; and 3) transport mechanism has a functional relevance with sodium homeostasis.

In the two first sections of this review, we summarize the general properties and the renal specificities of Na⁺-K⁺-ATPase and of intracellular signaling pathways. The three following parts are devoted to the hormonal regulation of cation transport in proximal tubule, thick ascending limb of Henle's loop, and collecting duct. For each structure, the cellular and molecular mechanisms of sodium transport are analyzed before discussing the actions of the main hormones. For each hormone, the general physiological response is first described, and then the regulation of Na⁺-K⁺-ATPase and other transporters is analyzed in terms of intrinsic changes in properties and of signaling mechanisms.

	II. SODIUM-POTASSIUM-ADENOSINETRIPHOSPHATASE IN SODIUM TRANSPORT ALONG THE RENAL TUBULE

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Epithelial cell layers separate compartments of distinct compositions and ensure transfer of water and solutes between them. The serosal compartment, in equilibrium with blood plasma, is characterized by the constancy of its composition. In contrast, the composition of the mucosal compartment varies greatly from one epithelium to another and with time. Epithelial cells are characterized by their functional polarization, since their apical membrane facing the mucosal compartment has receptors as well as transport and permeability properties distinct from their basolateral membrane bathed by the serosal compartment. This polarity is maintained by targeting to and/or withdrawal of newly synthesized proteins from a specific cell pole, and by prevention of planar diffusion of membrane constituents between the apical and basolateral domains by specific proteins located at the intercellular junctional complexes (115, 131).

In renal tubular cells, as in all sodium-reabsorbing epithelia, Na⁺-K⁺-ATPase is exclusively located in the basolateral membrane (723), the infoldings of which are closely surrounded by mitochondria. In contrast, the sodium gradient generated by Na⁺-K⁺-ATPase between intra- and extracellular compartments is mainly dissipated across the apical membrane. A net transfer of sodium from mucosal toward serosal compartment results from this architectural organization. Quantitatively, net sodium reabsorption is the major function of renal Na⁺-K⁺- ATPase, and a close relationship exists between the abundance of Na⁺-K⁺-ATPase and the sodium reabsorption capacity of the different segments of nephron (323). In humans, kidneys reabsorb over 600 g sodium/day and utilize over 2 kg of ATP for this process. Accordingly, kidney cells are rich sources of Na⁺-K⁺-ATPase; they contain up to 50 million pumps per cell (248) compared with a few hundred to a few thousand pumps in nonpolarized cells.

Renal Na⁺-K⁺-ATPase energizes not only sodium reabsorption, but also the secondary active transport (reabsorption or secretion) of large amounts of a wide variety of substances, including other ions and uncharged solutes. Indeed, passive entry of sodium into the cell is often coupled to the transport of other solute(s) at the level of symport or antiport systems. In turn, transport of charged solutes generates transmembrane voltage and concentration gradients that serve as driving force for passive electrolyte movements through ion channels. Transcellular movements of solutes may also generate a transepithelial potential difference that drives ion movements along the paracellular pathway, especially in leaky epithelia.

In summary, Na⁺-K⁺-ATPase can be considered as an energetic transducer that converts metabolic energy into rapidly mobilizable ionic solute gradients. Despite its ubiquitousness and its quantitative prevalence in kidney epithelial cells, it is worth recalling that Na⁺-K⁺-ATPase and sodium and potassium gradients are not a universal source of energy. This function may also be achieved by H⁺-ATPase and proton gradient in most lower eukaryotes, but also in some cells of higher eukaryotes, such as the intercalated cells of the collecting duct.

Some 40 years ago, 1997 Nobel Prize winner J. C. Skou (748) first reported that microsomal membrane fractions from crab nerve contain an ATP-hydrolyzing activity stimulated by concentrations of sodium and potassium usually found in intracellular and extracellular fluids, respectively (747). This requirement for both sodium and potassium ions remains the fundamental characteristic of Na⁺-K⁺-ATPase.

Na⁺-K⁺-ATPase activity is stimulated by sodium (acting at the cytosolic face of the membrane) with an apparent mean affinity constant (K_0.5) in the 5-15 mM range in the presence of 5-10 mM K⁺, and under these conditions the maximum velocity (V_max) is achieved with 60-100 mM of sodium. Because intracellular sodium concentration is in the 5-20 mM range, Na⁺-K⁺-ATPase works well below its V_max in intact cells. Thus any increase in intracellular sodium concentration stimulates Na⁺-K⁺-ATPase activity which, in turn, pumps more sodium out of the cell and thereby contributes to restore the initial intracellular sodium concentration. Conversely, any decrease in intracellular sodium concentration slows down the pump and participates in maintaining cellular homeostasis. This autoregulatory process is highly efficient because sodium activation of Na⁺-K⁺-ATPase displays a marked positive cooperativity; thus small variations of sodium concentration around the K_0.5 induce large variations of Na⁺-K⁺-ATPase activity. In addition, any regulatory process that alters the sodium affinity of Na⁺-K⁺-ATPase also alters the pump activity. From the extracellular side, Na⁺-K⁺-ATPase is stimulated by potassium with an apparent Michaelis constant (K_m) in the millimolar range (0.5-1.5 mM). Thus extracellular potassium is not rate limiting for ATPase activity, except in the case of severe hypokalemia.

The requirement for intracellular sodium is almost absolute except for lithium, which is transported (although at a slower rate than sodium) by human erythrocytes and kidney cells. This has no physiological relevance because lithium concentration in body fluids is very low. The selectivity for potassium is less strict since it can be replaced by rubidium and ammonium with almost similar affinities and efficiencies. Na⁺-K⁺-ATPase-mediated transport of ammonium instead of potassium into the cells has a physiological relevance in the kidney medulla since it participates to the recycling of ammonium. Transport of rubidium by Na⁺-K⁺-ATPase has no physiological significance, but use of rubidium as a potassium surrogate proved to be a precious tool for studying the sodium pump. Indeed, the radioactive isotope ⁸⁶Rb⁺ is much easier to handle in the laboratory than ⁴²K⁺ because it has a much longer radioactive half-life (18 days vs. 12 h).

The energy necessary to move sodium and potassium against their transmembrane electrochemical gradients is provided by the hydrolysis of the "energy-rich" ATP molecule, as other nucleotides triphosphate are hydrolyzed at much slower rates. The true substrate of Na⁺-K⁺-ATPase is the ATP-Mg complex, but the dependency on magnesium is not absolute because other divalent cations (manganese, cobalt) can substitute for magnesium. However, most divalent cations, in particular calcium, inhibit ATPase activity.

For each ATP molecule hydrolyzed, Na⁺-K⁺-ATPase moves two potassium ions into the cell and three sodium ions out of the cell. An important consequence of this 3Na⁺:2K⁺ stoichiometry is the electrogenicity of Na⁺-K⁺-ATPase and therefore its dependence on membrane potential (see below).

As with all P-type ATPases, Na⁺-K⁺-ATPase is transiently phosphorylated during its activation. P-type ATPases are also called E₁-E₂ ATPases because they exhibit two main conformation states that can be either unphosphorylated (E₁ and E₂) or phosphorylated (E₁-P and E₂-P). The two conformation states of Na⁺-K⁺- ATPase are characterized by their respective affinities for sodium, potassium, and ATP and by the accessibility of the cationic sites at the intracellular or extracellular sides of the membrane: E₁ conformation confers a high affinity for ATP and sodium and a low affinity for potassium, both cation sites being accessible from the intracellular side, whereas under the E₂ conformation, the cation sites are accessible from the outside and display low affinity for sodium and high affinity for potassium. Na⁺-K⁺-ATPase cycles through these different conformations according to the so-called Albers-Post model (Fig. 1). ATP, magnesium, and sodium bind to E₁ on the intracellular side of the pump, allowing phosphorylation of E₁ (E₁-P) and "occlusion" of sodium ions that are no longer accessible from either side of the membrane. After release of ADP, the exergonic transconformation of E₁-P to E₂-P occurs and promotes the extracellular delivery of sodium and the binding of extracellular potassium. This latter process induces dephosphorylation of E₂-P and potassium occlusion. Spontaneous reversion to E₁ releases potassium inside the cell, completing the reaction cycle (reviewed in Ref. 449).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Catalytic cycle of Na+-K+-ATPase. This models shows the transition between the main conformational forms of Na+-K+-ATPase catalytic subunit (E1, E1-P, E2-P, and E2) with either accessible or occluded cation binding sites. It also indicates the sites of action of Na+-K+-ATPase inhibitors vanadate and ouabain.

A) VANADATE. Vanadate acts as a structure analog of phosphate to inhibit all P-type ATPases through binding to their phosphorylation site and blockade under their E₂ configuration (Fig. 1). Although vanadate was initially considered as a putative physiological modulator of Na⁺-K⁺-ATPase (130), this now appears unlikely because the redox state prevailing within cells reduces vanadate to inactive vanadyl.

B) DIGITALIS GLYCOSIDES. Digitalis glycosides are natural and potent inhibitors of Na⁺-K⁺-ATPase (710) used in therapy as well as in the laboratory. Ouabain (G-strophantin) is generaly used in vitro because of its better, although limited, water solubility, whereas digoxin is the most widely used digitalic in therapy. Ouabain binds to an extracellular domain of Na⁺-K⁺-ATPase under its E₂ conformation and decreases its affinity for potassium, and vice versa, as a competitive inhibitor (Fig. 1). As a clinical counterpart, digitalic poisoning is more severe in hypokalemic patients. Through impediment of potassium binding, ouabain prevents the dephosphorylation of the enzyme and the associated transconformation from E₂ to E₁. The affinity of Na⁺-K⁺-ATPase for ouabain varies within a wide range of concentrations (from nM to mM) between species (rats, mice, and Bufo marinus are rather resistant to ouabain) and between organs or cells from a given species (kidneys being less sensitive to ouabain than brain and heart). These differences are accounted for in part by the molecular heterogeneity of Na⁺-K⁺-ATPase (see below).

For over 40 years, ouabain has been considered as a highly specific inhibitor of Na⁺-K⁺-ATPase. In particular, despite its functional and structural similarities with Na⁺-K⁺-ATPase, gastric H⁺-K⁺-ATPase is not sensitive to ouabain. However, it is now established that ouabain also inhibits several nongastric forms of H⁺-K⁺-ATPase (431).

C) PALYTOXIN. Palytoxin, a nonprotein toxin produced by a marine coelonterate, not only inhibits Na⁺-K⁺- ATPase but transforms it into a sodium channel (358). The tumor-promoting activity of palytoxin is related to its ability to increase intracellular sodium by this mechanism (482).

Purification (438) and molecular cloning (456, 615, 730, 734, 735, 736) have shown that Na⁺-K⁺-ATPase consists of two main subunits ( and ) that are associated in a 1:1 molar ratio.

The -subunit (~1,000 amino acid residues and 110 kDa) displays all the functional properties described above (binding sites for sodium and ATP and phosphorylation site on the cytoplasmic domain, and binding sites for potassium and ouabain on the extracellular domain) and is therefore considered as the catalytic subunit. It consists of 10 membrane-spanning domains (M₁-M₁₀) with intracellular NH₂- and COOH-terminal domains and a long M₄-M₅ intracytoplasmic loop (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Topology of the Na+-K+- ATPase -subunit and localization of the phosphorylated amino acids. The Na+-K+-ATPase -subunit displays 10 membrane-spaning domains with intracytoplasmic NH2 and COOH termini. The currently identified amino acids phosphorylated by protein kinase C (PKC) and tyrosine kinases (TK) are located in the extreme NH2 terminus of the -subunit. Both Tyr-10 and Ser-16 are conserved in all cloned 1-subunits, whereas Ser-23 is specific of the rat 1-subunit. The protein kinase A (PKA) phosphorylation site is located at Ser-943 into the M8-M9 intracellular loop. Also indicated is the Asp residue within the M4-M6 large intracellular loop that is phosphorylated during the catalytic cycle of the pump.

Site-directed mutagenesis of amino acid residues and covalent chemical modifications of the -subunit have permitted the identification of functionally important amino acids and domains (reviewed in Ref. 396). The ATP binding domain and the phosphorylation site are located into the long M₄-M₅ cytoplasmic loop; their highly conserved sequence is a molecular signature of P-ATPases. The M₄, M₅, and M₆ transmembrane domains likely constitute the cation occlusion site and the ion pore. The intracellular NH₂-terminal domain might play a role in cation gating. M₁-M₂ as well as part of the M₃-M₄ ectodomains are involved in ouabain binding. The M₇-M₈ ectodomain is the main site of interaction with the -subunit. Finally, several intracellular amino acid residues are phosphorylation sites for protein kinases (see below).

The -subunit is smaller (~300 amino acids) and displays a single membrane-spanning domain and a large extracellular domain with several N-linked glycosylation sites. This ectodomain is responsible for the interaction with -subunit. Although the -subunit has no enzymatic or transport activity, its association with an -subunit is an absolute requirement for ATPase and pump activities: it allows the folding of newly synthetized -subunits and their targeting from the endoplasmic reticulum to the plasma membrane, as well as the stabilization of -subunits within the membrane (328, 330, 828).

Na⁺-K⁺-ATPase may also contain a -subunit (53 amino acids, ~10 kDa), which was first recognized as copurifying with - and -subunits (296). More recently, this -subunit has been cloned (554); its mRNA is abundantly expressed in kidney and at lower level in other epithelia but is absent in other tissues. Thus, conversely to the - and -subunits, the -subunit is not an absolute requirement for functional Na⁺-K⁺-ATPase although, when present, it is an integral part of Na⁺-K⁺-ATPase. The -subunit contains a single transmembrane domain with an extracellular NH₂ terminus. Expression in Xenopus oocyte indicates that the -subunit reaches the cell membrane only when associated with the -complex (in a 1:1:1 stoichiometry) (69). Functionally, coexpression of -subunit with - and -subunits was described as modifying the voltage sensitivity of potassium activation (69), decreasing the affinity of the pump for ATP (805, 806) as well as for sodium and potassium (28).

Despite canonical characteristics, the Na⁺-K⁺- ATPase is functionally highly heterogeneous. Molecular cloning and expression of several isoforms of Na⁺-K⁺-ATPase catalytic subunits provided some molecular basis for this heterogeneity. As yet, four genes encoding Na⁺-K⁺-ATPase -subunits and three genes encoding -subunits have been cloned from mammals (reviewed in Ref. 396). In addition, two splice variants of the -subunit (₁ and ₂) have been recently characterized in rat kidney (483). The ₁-subunit has the amino acid sequence predicted by the published cDNA sequence, whereas the ₂-subunit contains a different sequence of its 7 NH₂-terminal amino acids and is acetylated on its first methionine. Experiments in which specific - and -isoforms were coexpressed in heterologous cellular systems indicate that all types of -dimers tested are functional. However, it remains to be demonstrated that all the combinations between different - and -isoforms are functionally expressed in normal cells. The following discussion on the properties of these different isoforms is focused around -subunits because they condition the main properties of the holoenzyme.

Sequence comparison between the different -isoforms in a given species reveals a very high degree of conservation, suggesting the existence of a common ancestor gene. The four isoforms of Na⁺-K⁺-ATPase -subunit (₁-₄) are differentially expressed among tissues: the ₁-isoform is ubiquitous and is the most abundant, if not the only, form in the kidney; the ₂-isoform is predominantly expressed in heart, skeletal muscle, and brain; the ₃ is expressed in neural tissue and ovary; whereas expression of the ₄-isoform is restricted to testis (786, 895).

In rat, it is well established that -subunit isoforms endow different affinities for cardiac glycosides: ₃ [dissociation constant (K_D) 2 nM] > ₂ (K_D100 nM) > ₄ (K_D300 nM) > ₁ (K_D1 mM) (602, 648, 895). The very low affinity of rat ₁-isoform for ouabain mainly results from the presence of a positively charged arginine residue and a negatively charged aspartate residue at the two ends of the first (M₁-M₂) ectodomain of the -subunit (649). Such differences in ouabain sensitivities are not found in all species; in humans, for example, the ₁-₃ isoforms display similar affinities for ouabain (190).

Isoforms of Na⁺-K⁺-ATPase -subunits may also differ by their voltage dependence. The equilibrium potential of Na⁺-K⁺-ATPase (i.e., the membrane potential for which the energy necessary to move three sodium and two potassium ions across the cell membrane equals the free energy of hydrolysis of one molecule of ATP) is around 280 mV. From that membrane potential, at which the pump cannot work, the pumping rate of Na⁺-K⁺-ATPase should theoretically increase as the membrane depolarizes to 0 mV and to positive potentials. Such relationships between membrane potential and Na⁺-K⁺-ATPase pumping rate (as evaluated by the pump current) were experimentally observed within a wide range of membrane potentials (from 100 to + 50 mV) in excitable cells (which mainly contain the ₁- and ₃-isoforms) such as axons (660) and myocytes (318). However, in renal epithelial cells (which mostly contain the ₁-isoform), the pump current does increase with membrane potential within the 175 to 75 mV range, but reaches a plateau at higher physiological potentials (75 to 25 mV) (397). Whether these distinct behaviors are intrinsic properties of the distinct isoforms originating in these two types of tissues or result from the specific cellular environment remains unknown. The marked voltage dependency of Na⁺-K⁺-ATPase in excitable cells is teleologically sound, since stimulation of the pump during membrane depolarization facilitates the recovery of intracellular sodium concentration during action potential. Also physiologically sound is the absence of such regulation in epithelial cells since their membrane potential does not vary much.

Thus the expression of functionally distinct isoforms of the Na⁺-K⁺-ATPase -subunit in specific tissues may have physiological consequences. In addition, these properties might be modulated by the association with different isoforms of -subunit and/or the absence or presence of the different splice variants of the -subunit.

An emerging but important regulatory mechanism for Na⁺-K⁺-ATPase activity in intact cells is phosphorylation by protein kinases. Phosphorylation of the Na⁺-K⁺- ATPase -subunit has been first recognized using purified preparations of Na⁺-K⁺-ATPase incubated in the presence of protein kinase C (PKC) or protein kinase A (PKA) (83, 283, 522). PKA- and PKC-mediated phosphorylation of Na⁺-K⁺-ATPase -subunit has been subsequently revealed in tissue homogenates (167) and in intact cells (66, 67, 86, 105, 134, 135, 295, 496, 529, 555, 616).

A single PKA phosphorylation site has been mapped to Ser-943 (Fig. 2) located in a typical PKA consensus site conserved in all cloned Na⁺-K⁺-ATPase -subunits (66, 283, 294). In addition, two PKC phosphorylation sites are currently identified (Fig. 2). The first one, located at Ser-16, is conserved among all cloned ₁-subunits and lies within an unusual PKC phosphorylation motif (66, 68). The second one, located at Ser-23, is found only in the rat ₁-subunit and is comprised of a typical PKC consensus site within the lysine-rich cluster of the NH₂-terminal domain of the ₁-subunit (68, 73, 284). Finally, phosphorylation of the Na⁺-K⁺-ATPase ₁-subunit at Tyr-10 (Fig. 2) has been recently identified (276), similarly to the closely related gastric H⁺-K⁺-ATPase -subunit (809). It should be mentioned that phosphorylation of the identified sites does not account for the whole basal phosphorylation of the Na⁺-K⁺-ATPase ₁-subunit (66, 68). These findings, together with the identification of threonine phosphorylation of rat ₁-subunit in intact cells (276), indicate that an additional phosphorylation site(s) remains to be identified.

The functional effects of serine phosphorylation of the -subunit are still highly debated. Results obtained in transfected COS-7 cells have suggested that PKA phosphorylation of the -subunit has an inhibitory effect on Na⁺-K⁺-ATPase activity (294). However, in vitro PKA phosphorylation of shark rectal gland Na⁺-K⁺-ATPase stimulates its activity, whereas the activity of the pig kidney enzyme is unchanged under similar conditions (186). It should be mentioned that in native rat kidney epithelial cells, PKA phosphorylation of the Na⁺-K⁺-ATPase is associated with stimulation of its activity (135, 467).

Similarly, in response to PKC phosphorylation of its -subunit, Na⁺-K⁺-ATPase activity was either stimulated (134, 628, 835), inhibited (73, 166, 168, 835), or unchanged (77, 285). These discrepancies observed in intact cells may be accounted for in part by the presence of both indirect effects of PKC phosphorylation such as internalization of active Na⁺-K⁺-ATPase units (165) and direct effects of phosphorylation such as an increase in apparent affinity for sodium (276). This later effect of phosphorylation of Na⁺-K⁺-ATPase -subunit was recently demonstrated using COS-7 cells stably transfected with either wild-type or Ser-16 (the ubiquitous PKC phosphorylation site) mutant Na⁺-K⁺-ATPase ₁-subunits: 1) phorbol esters increased the apparent sodium affinity of wild-type Na⁺-K⁺-ATPase; 2) when nonspecific increase in fluid-phase endocytosis was prevented, mutation of Ser-16 prevented the stimulatory effect of phorbol esters on the transport activity of Na⁺-K⁺-ATPase; and 3) mutant ₁-subunits in which Ser-16 was substituted by an acidic residue (Asp or Glu) mimicking constitutive phosphorylation exhibited an increased apparent sodium affinity (276). This effect of Ser-16 phosphorylation on the apparent sodium affinity of Na⁺-K⁺-ATPase is in agreement with the results of Logvinenko et al. (515), who showed that in vitro phosphorylation of purified Na⁺-K⁺-ATPase by PKC shifts the conformational equilibrium of the Na⁺-K⁺-ATPase toward the E₁ conformation, i.e., the conformation displaying high affinity for sodium (see sect. IIA1). These observations are also consistent with earlier studies showing that the ₁-subunit NH₂-terminal domain is involved in conformational changes of the enzyme. Indeed, tryptic cleavage of the ₁-subunit occurring between Lys-30 and Glu-31 (440), or truncation of the NH₂ terminus by site-directed mutagenesis (854, 880), displaces the E₁-E₂ conformational equilibrium toward the E₁ conformation through stimulation of potassium deocclusion (880), and thereby may account for the increased apparent sodium affinity of Na⁺-K⁺-ATPase (440). This recent report (276) agrees with a growing number of studies documenting a stimulation of Na⁺-K⁺-ATPase activity in response to PKC activation (277, 356, 393, 530, 628), but contrasts with a few others (73, 285). It should be stressed, however, that these last two studies (73, 285) focused on the role of the additional rat-specific Ser-23 phosphorylation site and not on that of the ubiquitous Ser-16 phosphorylation site. These two phosphorylation sites might be targets for different PKC isozymes and/or produce different physiological effects. Indeed, phosphorylation of Ser-23 per se does not alter Na⁺-K⁺-ATPase activity (165, 285) but promotes endocytosis of the Na⁺-K⁺ pump in proximal tubule and in opossum kidney (OK) cells (165, 166). This hypothesis is supported by recent data indicating that 1) in OK cells, the opposite effects of phorbol esters and dopamine on the transport activity of Na⁺-K⁺-ATPase rely on classical PKC- and atypical PKC- activation, respectively (79); and 2) the transport activity of rat ₁- complexes expressed in Xenopus oocytes is inhibited while that of the endogenous Xenopus₁- complexes, which were previously shown to be exclusively phosphorylated on Ser-16 (68), are stimulated by injection of purified rat PKC (835). In addition, secondary regulatory mechanisms that may be cell specific and/or brought about by experimental conditions, such as oxygen (81, 277) or calcium (161) availability, may also explain some discrepancies.

In response to the activation of receptor tyrosine kinases, i.e., insulin, insulin-like growth factor I (IGF-I), or epidermal growth factor (EGF) receptors, stimulation of Na⁺-K⁺-ATPase activity is almost always reported (178, 209, 266, 279, 280, 531, 544, 766). These observations are consistent with the recent identification of Tyr-10-dependent stimulation of Na⁺-K⁺-ATPase activity in native and cultured renal proximal tubule epithelial cells (278).

B.  Na⁺-K⁺-ATPase Along the Nephron

1.  Anatomic and topographic segmentation of the nephron

Figure 3 schematically depicts the topographical organization and the axial segmentation of the rat nephron and lists the abbreviations used in this review for the different nephron segments.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Topology of the main nephron segments and sites of action of hormones controling sodium transport. Schematical representation of a nephron with its successive portions: 1) glomerulus; PCT, proximal convoluted tubule; PST, proximal straight tubule; 2) thin descending limb of Henle's loop; 3) thin ascending limb; MTAL, medullary thick ascending limb of Henle's loop; CTAL, cortical thick ascending limb; 4) macula densa; 5) distal convoluted tubule; 6) connecting duct; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct. For the three structures analyzed in this review (proximal tubule, thick ascending limb of Henle's loop, and collecting duct), we mentioned the main hormones or factors that stimulate () and inhibit (|) sodium reabsorption: ANG II, angiotensin II (low and high referring to pico- and micromolar concentrations); adr, adrenergic agonists; AVP, arginine vasopressin; PTH, parathyroid hormone; GC, glucocorticoids; MC, mineralocorticoids; PGE2, prostaglandin E2; ET, endothelin; ANP/Urod, atrial natriuretic peptide and urodilatin; PAF, platelet-activating factor; BK, bradykinin.

The proximal tubules extend from the glomeruli down to the thin segments, at the junction between the outer and inner stripes of the kidney outer medulla. Its apical cell border is characterized by a well-developed brush border made of densely packed microvilli. The basolateral plasma membrane forms deep infoldings that are in close contact with mitochondria. Both apical microvilli and basolateral membrane infoldings considerably increase the membrane surface area available for transport. Intercellular cell junctions are shallow, and the epithelium is leaky. The proximal tubule is usually subdivided in three successive portions on a morphological basis: S₁ includes the initial and mid proximal convoluted tubule (PCT), S₂ includes the late PCT and the cortical portion of the proximal straight tubule (PST), and S₃ consists of the outer medullary PST. In most species but rat, the cell size, the density of apical microvilli, and the height of brush border and basolateral infoldings decrease from S₁ to S₃, and along with these morphological changes, the transport capacity of the proximal tubule also decreases from S₁ to S₃.

The thin segments of the loop of Henle extend from the end of PST up to the junction with thick ascending limbs. Despite their importance for urine concentration by a countercurrent mechanism, they are exclusively the site of passive solute and fluid exchanges. Accordingly, their Na⁺-K⁺-ATPase activity is very low (see below), and therefore, they are not considered in the following sections.

The thick ascending limb of Henle's loop (TAL) extends from the junction between the inner and outer medulla (where the thin segments end) up to the macula densa, or a few micrometers beyond, in the superficial cortex. It therefore includes a medullary and a cortical portion (MTAL and CTAL, respectively). On the basis of morphological criteria, TAL appears as made of a single type of cell that displays deep basolateral membrane infoldings surrounding numerous mitochondria. Apical membrane forms only few and short microvilli, and the junctional complexes are numerous and of the shallow type. Functionally these intercellular junctions are permeable to solutes but highly impermeable to water.

The distal convoluted and connecting tubules (DCT and CNT, respectively) complex, which extends from the macula densa to the first branching with another tubule, represents an heterogeneous portion from morphological, functional, and biochemical points of view. It consists of three cell types (DCT cells, connecting cells, and intercalated cells), the distribution of which along the DCT and CNT varies with species. In most species, however, the three cell types are present along most of the length of this nephron portion. Despite the functional and pharmacological importance of the DCT/CNT segments, in particular for calcium reabsorption and as the site of action of thiazide diuretics, their shortness and their cellular heterogeneity have precluded the determination of the molecular mechanism underlying the regulation of their transport properties. Therefore, this regulation is not discussed in the following sections.

Collecting ducts constitute the last segment of the nephron. They extend throughout the kidney, from the outermost cortex to the tip of the papilla, and therefore, they encounter surroundings of different compositions. On the basis of topographical criteria, the collecting duct is usually subdivided into three successive portions: the cortical collecting duct (CCD), the outer medullary collecting duct (OMCD), which is itself subdivided into outer stripe and inner stripe subsegments (OMCD_o and OMCD_i, respectively), and the inner medullary collecting duct (IMCD) also subdivided into three subsegments of equal length (IMCD₁, IMCD₂, and IMCD₃). The CCD and OMCD are made of two distinct cell types, namely principal, or light, cells (accounting for 60-65% of whole cells) and intercalated, or dark, cells (accounting for the remaining 35-40% cells). Intercalated cells are further subdivided into two subtypes called type A and type B intercalated cells (or - and -cells). Principal cells are characterized by a deeply invaginated basolateral membrane, the infoldings of which are closely associated with mitochondria, and a rather smooth apical membrane with few blunt microvilli and a single central cilium. Intercalated cells are characterized by a great number of mitochondria. Type A cells display extensive apical microplicae, numerous subapical tubulovesicular structures, and a rather nonextensive basolateral membrane. In contrast, type B cells display fewer apical microplicae, the tubulovesicular structures are scattered throughout the cell, and the basolateral membrane is extensive. In fact, these are archetypal descriptions of types A and B intercalated cells, as one can distinguish intermediate subtypes of intercalated cells featuring only a few of those structural properties: intercalated cells can evolve from one phenotype to the other in response to different stimuli, and intermediate subtypes may correspond to evolving cells (8). Under normal conditions, there is an axial gradient in the relative proportion of the two subtypes of intercalated cells along the CCD and OMCD: type B cells are preponderant in the most cortical portion of the collecting duct (25-28% B cells vs. 10-12% A cells), whereas they almost disappear at the transition between OMCD_o and OMCD_i.

The OMCD_i and IMCD₁ consist of principal cells and type A intercalated cells. The proportions of intercalated cells decrease from 35-40% at the transition between OMCD_o and OMCD_i to 10% at the IMCD₁-IMCD₂ transition. The morphology of principal cells also varies from the most cortical regions of CCD down to the innermost region of IMCD₁. The most prominent aspect of axial changes is the decrease in size of the basolateral membrane and in the density of intracellular organelles, which correlates with a decrease in transport capacity.

IMCD₂ and IMCD₃ are made of IMCD cells that appear structurally homogeneous, eventhough there might be several functionally distinct subtypes of IMCD cells.

Although early approaches were based on isolation of well-defined portions of renal tubule from freeze-dried kidney sections (718), all the techniques presently used apply to nephron segments microdissected from fresh tissue. Moderate hydrolysis of kidney interstitium with collagenase allows the isolation of large numbers of samples from all the nephron subsegments. The main limitation of this approach results from the small size of microdissected samples: a 1-mm-long nephron segment accounts for ~300-400 cells, 100-250 ng of proteins, 3-5 ng RNAs, and 60-100 pg poly(A) RNAs. Nonetheless, techniques are now available for quantifying the enzymatic and transport activity of Na⁺-K⁺-ATPase, the number of catalytic units, as well as the amount of protein and of mRNA coding its different subunits at the level of single or homogeneous populations of nephron segments.

The enzymatic (ATP-hydrolytic) activity of Na⁺-K⁺-ATPase can be measured at the level of single nephron segments by monitoring the rate of hydrolysis of exogenous ATP. The amplification factor required by the small size of the sample is provided either by using radioactive ATP (224) or by enzymatically coupling the production of ADP to the generation of a fluorescent metabolite (323, 633). Whatever the method used, the assay needs to be carried out on broken or permeabilized cells to permit the access of exogenous ATP to intracellular catalytic sites. The Na⁺-K⁺-ATPase activity should be discriminated from other ATPase activities present in the nephron segments on the basis of its sodium and potassium dependence rather than its sensitivity to ouabain, since this drug may inhibit other ATPases (nongastric H⁺-K⁺- ATPase) potentially present in the tubular sample. Alternatively, it is possible to use specific inhibitors of these ouabain-sensitive H⁺-K⁺-ATPases (such as Sch 28080) to circumvent this contamination. Measurement of Na⁺-K⁺-ATPase activity allows us to define and control the concentration of substrates during the assay, and thereby to determine the kinetical parameters of the enzyme, in particular its V_max and K_0.5 for cations. As a counterpart, permeabilization of cell membranes entails the loss of regulatory parameters such as the transmembrane voltage, the membrane limitation to potassium recycling, or intracellular diffusible regulatory molecules. Also, Na⁺-K⁺-ATPase activity does not inform about the in vivo pumping activity of Na⁺-K⁺-ATPase, as the latter is highly dependent on intracellular sodium concentration.

In nonpermeabilized nephron segments, the activity of Na⁺-K⁺-ATPase can be determined through its pumping capacity by measuring ouabain-sensitive rubidium uptake under initial rate conditions (163). Although this method is widely used in a great variety of tissues, it has two main pitfalls. The first one, which results from the nonspecificity of ouabain (see above), can be easily circumvented by using Sch 28080 in the assay to abolish the activity of ouabain-sensitive H⁺-K⁺-ATPases. More importantly, ouabain-sensitive rubidium uptake is calculated as the difference between the rates of intracellular accumulation of rubidium measured in the absence and presence of saturating concentrations of ouabain, respectively. Such a calculation implies that Na⁺-K⁺-ATPase-independent rubidium intake is similar in the absence and presence of ouabain. This is very unlikely, since inhibition of Na⁺-K⁺-ATPase by ouabain abolishes the concentration gradient of rubidium across cell membranes and may thus increase the driving force for passive, Na⁺-K⁺-ATPase-independent rubidium movements. Thus ouabain-insensitive rubidium uptake probably overestimates Na⁺-K⁺-ATPase-independent rubidium uptake, and therefore, ouabain-sensitive rubidium uptake underestimates Na⁺-K⁺-ATPase activity. However, because ouabain-insensitive rubidium uptake in renal tubule cells represents a minute fraction of the total rubidium uptake (163), this underestimation might be limited. An additional technical difficulty of this measurement in isolated nephron segments results from their huge Na⁺-K⁺-ATPase activity: the kinetics of rubidium uptake is fast, and measurement under initial rate conditions must be performed within 0.5-1 min. Changes in rubidium uptake reflect alterations of the V_max of the pump and/or changes of its efficiency brought about by changes in intracellular concentration of sodium and/or in affinity for sodium.

Despite its importance, the regulation of the pump affinity for intracellular sodium concentration remains very difficult to evaluate in intact nephron segments, since this requires the precise clamping and monitoring of intracellular sodium concentration. An elegant method has been proposed (97) in which the rate of sodium efflux is monitored as a function of time in nephron segments initially loaded with ²²Na⁺ by cold exposure in a potassium-free medium. Because the apical entry of sodium is blocked during the efflux study, i.e., the specific radioactivity of ²²Na⁺ remains constant, at any time one can calculate the sodium pumping rate of Na⁺-K⁺-ATPase from the rate of appearance of ²²Na⁺ in the superfusate and the intracellular concentration of sodium from the remaining quantity of ²²Na⁺. This allows the correlation of Na⁺-K⁺-ATPase functional activity in intact cells to intracellular sodium concentration. Unfortunately, this method has not been given the large use it deserves.

To elucidate whether changes in Na⁺-K⁺-ATPase activity are due to activation of preexisting units or induction of new ones, it is possible to quantify these units as well as the mRNAs encoding them. Quantification of Na⁺-K⁺-ATPase units is feasible on single nephron segments by measuring the specific binding of [³H]ouabain under saturating conditions (248). Contamination by ouabain-sensitive H⁺-K⁺-ATPase can be prevented by Sch 28080, which is a competitor of ouabain (119). Unfortunately, this method is hardly applicable to the rat because its kidney Na⁺-K⁺-ATPase displays a low affinity for ouabain. Alternatively, relative quantification can be made by Western analysis with specific antibodies against the different subunits of Na⁺-K⁺-ATPase (547). Although it requires pooling several tens of nephron segments in each sample, this approach is now routinely used. It is even possible to discriminate between plasma membrane pump and intracellular pools by biotinylation of membrane proteins and streptavidin precipitation before Western blotting analysis (136).

Finally, the transcripts of Na⁺-K⁺-ATPase subunits can be quantitated by quantitative RT-PCR (118, 818). Given the sensitivity of PCR and the abundance of renal Na⁺-K⁺-ATPase, this is feasible on very short portions of nephron (0.1 mm).

Measurements of Na⁺-K⁺-ATPase activity in microdissected segments of nephrons from different mammalian species have revealed the heterogeneity of distribution of this pump along the nephron (323, 454). In all species studied as yet, Na⁺-K⁺-ATPase activity is high in the TAL and DCT, intermediate in the proximal tubule (PCT and PST), relatively low in the collecting duct, and vanishingly low in the thin segments of Henle's loop. This distribution profile is paralleled by the number of Na⁺-K⁺-ATPase units determined either by [³H]ouabain binding (-complexes) (248) or by Western blotting (- and -subunits) (547).

From the number of specific [³H]ouabain binding sites and the V_max of Na⁺-K⁺-ATPase catalytic activity, one can calculate a molecular activity of ~2,000 cycles·ouabain binding sites¹·min¹ in all the nephron segments (248). This molecular activity is much lower than the 10,000 cycles·ouabain binding sites¹·min¹ reported for the Na⁺-K⁺-ATPase purified from kidney (439), suggesting the presence of cellular components that downregulate the enzyme activity in the cell.

With the assumption of a 1 ATP:2 K⁺ stoichiometry (see sect. IIA1), comparison of Na⁺-K⁺-ATPase activity and ouabain-sensitive rubidium uptake indicates that in intact tubular cells the pump is working at 20-30% of its maximum rate, which is consistent with measurements of intracellular sodium concentration and sodium affinity (163).

Despite technique availability, only one report, on rat OMCD, provides absolute quantification of Na⁺-K⁺- ATPase subunits mRNA expression (118). In this study, mRNAs for the ₁-subunit were greater than threefold more abundant than those coding for ₁-subunit, suggesting that the transcription of ₁-subunit mRNAs is the rate-limiting step in the regulation of Na⁺-K⁺-ATPase expression. Absolute quantification of mRNAs and of [³H]ouabain binding sites indicated that there are 24,000 Na⁺-K⁺-ATPase units per ₁-subunit mRNA, which suggests either a high efficiency of translation or a slow turnover rate of the protein.

The functional properties of Na⁺-K⁺-ATPase vary along the rabbit nephron: compared with proximal tubules and TAL, the collecting ducts display a higher affinity for ouabain (220) and for sodium (48). In addition, Na⁺-K⁺-ATPase activity is inhibited by a specific anti-₃-isoform monoclonal antibody in collecting ducts and by an anti-₁-isoform monoclonal antibody in proximal tubules and thick ascending limbs (46). In the rat nephron, two functional forms of Na⁺-K⁺-ATPase displaying different sensitivities to ouabain and to the anti-₁- and anti-₃- antibodies are coexpressed in each nephron segment (275).

However, despite some controversy (6, 176), most studies (162, 821) failed to demonstrate the presence of ₂- or ₃-isoforms in the rat nephron (₄ has not been looked but seems restricted to testis). The ₁₁ heterodimer is likely the exclusive Na⁺-K⁺-ATPase complex expressed in kidney tubules, and the functional axial heterogeneity of kidney Na⁺-K⁺-ATPase might result from cell-specific regulation. An interesting possibility would be that coexpression of the -subunits might be responsible for the observed differences (at least for sodium affinity).

	III. HORMONE SIGNALING ALONG THE NEPHRON

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A.  General Mechanisms of Hormone Signaling

1.  Receptors coupled to G proteins and adenylyl cyclase

Many peptide hormones, catecholamines, eicosanoids, nucleotides, and calcium ions act through binding to plasma membrane receptors coupled with specific multimeric G proteins. These G proteins are constituted by the association of a G-subunit with a G- and a G-subunit. Binding of a ligand to its cognate receptor induces a conformational change that is transmitted to the G protein, causing GDP release and GTP binding by the G-subunit, and promoting its dissociation from G-subunit complex. The free G-subunit and G-heterodimer each activate target effectors. The reaction is turned off by GTP hydrolysis and reassociation of the G protein subunits. Among the multiple targets of G protein subunits, adenylyl cyclase and phospholipase C- couple the binding of an agonist to its receptor with the modulation of PKA and PKC activity, respectively.

In the kidney, peptide hormones, e.g., parathyroid hormone and vasopressin, and catecholamines, e.g., epinephrine and dopamine, bind to G protein-coupled receptors and lead to the activation of adenylyl cyclases. Adenylyl cyclases are stimulated by G_s (795) but are either further stimulated (322, 793) or inhibited by G (453, 753, 796) according to their subtypes (Fig. 4). Other mediators such as angiotensin II or norepinephrine can decrease the generation of cellular cAMP through an inhibition of adenylyl cyclases by G_i subunits (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Schematic overview of the cAMP-PKA signaling pathway. Activation of cell-surface serpentine receptors (not shown) induces the dissociation of heterotrimeric G proteins into free -subunits and -subunits that may either activate or inhibit adenylyl cyclases (AC). The cAMP generated by adenylyl cyclases binds to the regulatory subunits (RI or RII) of PKA, allowing the dissociation of the catalytic subunits (C). The free catalytic subunits (C+) phosphorylate target membrane or cytosolic proteins. Subcellular targeting of PKA is mediated by interactions between A-kinase anchoring proteins (AKAPs) and the RII isoform of the regulatory subunit. Arrows indicate the direction of the signaling cascade and the resulting stimulatory (+) or inhibitory () effect on their targets.

Stimulation of adenylyl cyclase increases the intracellular concentration of cAMP leading to the activation of cAMP-dependent protein kinase (PKA) (Fig. 4). PKA is a heterotetramer consisting of a dimer of regulatory (R) subunits that maintains two catalytic (C) subunits in an inactive state. Upon binding of cAMP to the R subunits, active C subunits are released and can phosphorylate substrates. PKA phosphorylates serine or threonine residues located in consensus sites exhibiting the Arg-Arg-Xaa-Ser/Thr or Lys-Arg-Xaa-Xaa-Ser/Thr motifs. To date, three isoforms of C subunit (, , and ) and two isoforms of R subunits (RI and RII) have been identified in mammals. Some level of specificity of the PKA signal relies on the different affinities for substrates of C subunit isoforms as well as their binding properties to regulatory subunits (320). In addition, the RI and RII subunits exhibit different cAMP binding affinities and differential subcellular localization (203, 797). The type I PKA holoenzyme (containing RI) is predominantly cytoplasmic, whereas the type II PKA holoenzyme (containing RII) is mostly targeted to several subcellular compartments through binding to A-kinase anchoring proteins (AKAP) (203), conferring a further level of specificity. It is interesting to mention that some AKAP, e.g., AKAP79, can act as scaffolding proteins and also bind PKC isozymes (469) and protein phosphatase 2B (179).

Recent pieces of evidence suggest the presence of alternate PKA-independent cAMP signaling pathways. Two intracellular cAMP-binding proteins were cloned and shown to act as guanine nucleotide exchange factors for the small G protein Rap-1 (207, 457). Thus cAMP can activate the Rap-1 pathway independently of PKA activation. Interestingly, one of these two cAMP binding proteins (Epac or cAMP-GEF-I) is expressed at very high levels in the kidney (207, 457). Further studies are needed to identify the functional targets of the cAMP/Epac/Rap-1 pathway, in particular in kidney.

Termination of the signal is accounted for by activation of compartimentalized phosphodiesterases (169, 729) and desensitization of adenylyl cyclases and receptors (225, 354, 434, 516, 863).

PKCs constitute a superfamily of protein kinases comprising 11 isozymes. These isozymes are currently grouped into three families (Fig. 5) according to their sensitivities to physiological and pharmacological activators (511, 553). The most studied group is the conventional PKCs, which includes the -, I-, II-, and -isoforms. PKC-I and -II isoforms are generated by alternative splicing of the same gene. Conventional PKCs are activated by phosphatidylserine (PS) in a diacylglycerol (DAG)/phorbol esters and calcium-dependent manner. DAG and phorbol esters decrease the calcium concentration required for activation and dramatically increase PKC sensitivity to PS. PKCs , , , and  are members of the novel PKC family. These isozymes are calcium insensitive but remain activated by DAG and phorbol esters in the presence of PS. Finally, the members of the atypical PKC family, PKC- and PKC-, are insensitive to calcium, DAG, and phorbol esters, and their mode of activation is not clearly established. PKCs phosphorylate serine or threonine residues located in consensus sites exhibiting the Ser/Thr-Xaa-Lys/Arg motif. A first level of specificity is conferred by the cell-specific expression of PKC isozymes. For instance, the kidney expresses high levels of the classical PKC- but, in contrast to brain, - and -isozymes are undetectable (451, 612). The novel PKC- and - as well as the atypical PKC- are also expressed in the kidney cortex and medulla (451, 612). A second level of specificity is conferred by the differential pattern of PKC isoform activation in response to an agonist. In the kidney PCT, PKC-, -, and - are activated in response to phorbol esters, whereas only PKC- and - are activated in response to angiotensin II (451). The identification of PKC isozyme-specific anchoring proteins provides further specificity to the signal by targeting either active or inactive PKC to various subcellular compartments (505, 558).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Schematic overview of the PKC signaling pathway. Activation of cell-surface serpentine receptors (not shown) induces the dissociation of heterotrimeric G proteins into free -subunits and -subunits that may activate phospholipase C- (PLC-). Alternatively, growth factor receptors (GFRs) may be coupled to the PLC- isoform. PLCs generate diacylgylcerols (DAG) and inositol trisphosphate (IP3) that increases cytosolic calcium. The classical isoforms of PKC (cPKC) are activated by calcium and DAG, the novel isoforms of PKC (nPKC) are calcium insensitive but DAG activated, whereas the atypical PKC (aPKC) are both calcium and DAG insensitive. The current model for PKC targeting to specific subcellular compartments assumes that once activated, PKC isozymes translocate from receptors for inactive C-kinase (RICK) to receptors for activated C-kinase (RACK). Arrows indicate the direction of the signaling cascade and the resulting stimulatory (+)effect on their targets.

Insulin and many growth factors, e.g., IGF-I, EGF, and platelet-derived growth factor (PDGF), share the property to bind to receptors exhibiting an intrinsic tyrosine kinase activity (Fig. 6). Binding of the ligand to the receptor activates its tyrosine kinase activity and induces receptor autophosphorylation as well as phosphorylation of substrate proteins on tyrosine residues (458). It is generally admitted that growth factor receptors form homodimers either in the absence of ligand, e.g., PDGF receptor, or after ligand binding, e.g., EGF receptor, and that autophosphorylation results from the phosphorylation of one receptor monomer by the other (382). Because it is well established for EGF and PDGF receptors, tyrosine phosphorylation of the receptor creates specific binding sites for SH2 or PTB domains contained in many proteins involved in the subsequent steps of the signaling cascade (180, 455, 458, 499). These interactions, together with growth factor receptor aggregation, induce the focal formation of signaling complexes. In the insulin signaling pathway, tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) instead of the insulin receptor itself mediates the binding of signaling intermediates (875, 876). Receptor tyrosine kinases and IRS-1 can be associated with nonreceptor tyrosine kinases of the Src family (783, 824), phospholipase C- (474, 913), the regulatory p85 subunit of phosphatidylinositol 3-kinase (PIK) (35, 258, 452, 474), the adaptor proteins Grb2 (582, 857) or Shc (93, 357), and the tyrosine phosphatase SHPTP-2 (74, 488, 905). These protein-protein interactions may link receptor protein kinases to 1) a second round of tyrosine phosphorylation of specific target proteins by nonreceptor tyrosine kinases, e.g., c-Src (458, 614, 761); 2) PKC activation through activation of PLC- and PIK (205, 458, 568), as well as isoform-specific tyrosine phosphorylation, e.g., PKC- (205, 477) and PKC- (677); 3) PIK-dependent generation of phosphatidylinositol 3-products (132, 661) and activation of protein kinase B (297); and 4) Ras-dependent activation of extracellular regulated kinases (ERK) secondary to the binding of Sos (a guanine nucleotide exchange factor for Ras) to Grb2 and Shc (521, 650, 666) and activation of Raf-1 kinase by active Ras (877).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Schematic overview of growth factor receptor signaling. Once activated by ligand binding, GFRs dimerize and undergo autophosphorylation on specific tyrosine residues. Phosphotyrosines generate binding sites for SH2 and PTB domains. For instance, phosphorylated receptors can bind PLC-, the nonreceptor tyrosine kinase Src, the tyrosine phosphatase SHPTP2, the p85 subunit of the phosphatidylinositol 3-kinase (PI 3-K), and the adaptor proteins Grb2 and Shc. PLC-, Src, and PI 3-K link GFR to the PKC pathway, PI 3-K links GFR to the protein kinase B (PKB) pathway, and Grb2 links GFR, directly or through Shc binding, to Sos which activates Ras leading to Raf, MEK, and ERK activation. Arrows indicate the direction of the signaling cascade and the resulting stimulatory (+) effect on their targets.

Several mechanisms participate in the termination of the signal: dephosphorylation of receptors and signaling complexes by tyrosine phosphatases (174, 263, 777, 858), serine-threonine phosphorylation of receptors (265, 569, 807) and docking proteins, e.g., IRS-1 (626, 794), and internalization of activated receptors through clathrin-coated pits (133, 437, 754, 763, 883).

Traditionally, steroids, thyroid hormone, vitamins A- and D-derived hormones, and some fatty acids are thought to primarily alter the transcription of specific mRNAs and protein synthesis. This mechanism of action was initially proposed on the basis of the sensitivity of the functional actions of these agents to inhibitors of transcription and translation such as actinomycin D and cycloheximide (644). It also accounts for the latency and slow development of the cells responses to these agents. Later, this hypothesis was confirmed by cloning the cDNAs encoding intracellular receptors for these factors (61, 256, 312). Indeed, steroids and related agents bind to a superfamily of intracellular receptors that interact with regulatory elements of DNA, and thereby control the transcription of specific genes.

Steroid-thyroid-retinoid receptors consist of three major distinct domains: an immunogenic domain, a DNA binding domain, and a hormone binding domain. The DNA binding domain, which is the best conserved among different steroid receptors, consists of ~70 amino acids and contains two zinc finger structures (in which cysteines are coordinated by zinc), each followed by an -helix domain. These structures are important for recognition and binding to DNA and for dimerization of the receptor (256). The DNA binding domain recognizes specific target sequences on the DNA, called hormone response elements (HREs), which are very similar for the different receptors. For example, glucocorticoid, progesterone, androgen, and mineralocorticoid receptors recognize the same glucocorticoid response elements (GREs) with the consensus sequence 5'-NGGTACANNNTGTTCTN-3'. The hormone binding domain, a well-conserved region among distinct steroid receptors, consists of 250 amino acids forming an hydrophobic