10 Regulation of Receptor Function

10

Regulation of Receptor Function JEFFREY L. BENOVIC ROBERT J. LEFKOWITZ Howard Hughes Medical insiiiute Deparimenis of Medicine (Cardiology) and Biochemistry Duke University Medical Center Durham, North Carolina 27710

I. Introduction and Perspectives ................................. 11. The P-Adrenergic Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Heterologous Desensitization B. Homologous Desensitization .................................... 111. Rhodopsin .................................... IV. The Nicotinic Acetylcholine Receptor ............................... ................. V. The Receptors for EGF and Insulin . . . . . VI. Other Membrane Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .................

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Introduction and Perspectives

Receptors for hormones, drugs, and other biologically active molecules are crucially positioned to serve as important points of cellular metabolic regulation. They represent the critical locus of interaction of ligands with cells. Thus receptors control the responsiveness of cells to hormonal and pharmacological agonists or the entrance of key substances into cells via receptor-mediated endocytosis pathways. Mechanisms for regulation of receptor function have obvious potential for controlling these important cellular metabolic events. 319 THE ENZYMES, Vol. XVllI

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved

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The diverse and widespread role of phosphorylation in regulating the function of many enzymes is amply demonstrated by previous chapters in this volume. Recognition of enzyme regulation preceded appreciation of the role of protein phosphorylation in the regulation of receptor function. A key to progress in this area has been the ability to study the receptors for a wide variety of biologically active substances by techniques such as ligand binding, affinity chromatography, and photoaffinity labeling. This has permitted the study of the modification of receptor structure and function by phosphorylation. In this chapter we focus on the phosphorylation of several important plasma membrane receptors: the P-adrenergic receptor coupled to adenylate cyclase; rhodopsin, the archetypal “light” receptor of the rod outer segment; the nicotinic cholinergic receptor; the IgE receptor; and the transferrin receptor, which is involved in the internalization of iron-transferrin complexes. In addition, several cell surface receptors that possess tyrosine kinase activity (e.g., insulin and EGF receptors) are briefly discussed since they are dealt with in detail elsewhere in this volume. These specific cases provide examples of several mechanisms by which phosphorylation may regulate receptor function.

II. The f3-Adrenergic Receptor The P-adrenergic receptor is an ubiquitous plasma membrane glycoprotein that mediates catecholamine stimulation of the enzyme adenylate cyclase [reviewed in Refs. (1, 2 ) ] . This stimulation occurs via an agonist-induced, GTPdependent, interaction of receptor and N,, the stimulatory guanine nucleotide regulatory protein; N, can then directly activate adenylate cyclase leading to increased intracellular cAMP levels and subsequent activation of CAMP-dependent protein kinase. The P-adrenergic receptor is the only one of the adenylate cyclase-coupled receptors that has been purified and about which structural information is currently available. When isolated from mammalian sources it is visualized on SDSpolyacrylamide gel electrophoresis (SDS-PAGE) as a single polypeptide of M, 64,000. The receptor isolated from avian erythrocytes consists of two peptides, M, 40,000 and 50,000, present in variable proportions with the smaller peptide likely derived from the larger by proteolysis. Nonetheless both peptides appear to contain the intact P-adrenergic-ligand binding site. The amphibian erythrocyte P-adrenergic receptor has an apparent M, of 58.000. One of the striking features of the P-adrenergic receptor-adenylate cyclase system is that prolonged incubation of catecholamines with a cell leads to a diminution or blunting of the response to further challenge by the agonist. This process, termed “desensitization,” leads to reduced cAMP levels in the cell and consequently to a reduced cellular response to the hormone.

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Several different patterns of desensitization or refractoriness may be observed. In one type, generally termed “homologous desensitization,” exposure to a pagonist leads to diminished responsiveness only to subsequent stimulation by pagonists. The ability of other agonists to stimulate the adenylate cyclase through distinct receptors is preserved. In “heterologous desensitization” a more general blunting of responsiveness to other hormonal activators is also observed. In some situations even the actions of agents that bypass receptors in their stimulatory actions on the enzyme are reduced (e.g., NaF, forskolin). Heterologous desensitization appears to be modulated, at least in part, by CAMP, whereas homologous desensitization is not CAMPmediated. Considerable additional evidence, which has been recently reviewed elsewhere (3, 4), strongly suggests that different mechanisms are involved in homologous and heterologous desensitization. Homologous desensitization clearly seems to involve alterations in receptor activity whereas heterologous desensitization may be mediated by changes in the receptors or more distal components of the system. Homologous desensitization invariably involves sequestration or internalization of the receptors whereas heterologous desensitization involves receptor uncoupling from adenylate cyclase stimulation without receptor sequestration.

A. HETEROLOGOUS DESENSITIZATION Evidence has been presented that strongly implicates P-adrenergic receptor phosphorylation in the mechanism of heterologous desensitization. Stadel er al. (5) were the first to directly examine this question using turkey erythrocytes, a convenient model for the study of a heterologous form of desensitization. Using photoaffinity labeling techniques, it was found that the electrophoretic mobility on SDS-PAGE of (3-adrenergic receptors derived from desensitized turkey erythrocytes was different from that of receptors derived from control cells. The desensitized receptors appeared to migrate with decreased mobility. This alteration correlated with desensitization and could be mimicked by incubating the cells with cyclic nucleotide analogs. These findings were the first to suggest that phosphorylation of the p-adrenergic receptor might occur during desensitization since comparable alterations in electrophoretic mobility of other proteins after phosphorylation have been described (6, 7). In order to test this hypothesis, intact turkey erythrocytes were incubated with ’*Pi to label the intracellular ATP pool (8).Cells were then incubated with either buffer alone, agonist to promote desensitization or agonist plus antagonist to block the desensitization. The P-adrenergic receptors from the three groups were then partially purified by solubilization and affinity chromatography before characterization by SDS-PAGE. Figure 1 is an autoradiogram depicting such an experiment. Lanes 1 and 2 show [Iz5I]pABC (a p-adrenergic specific pho-

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FIG. 1. Autoradiogram of a SDS-PAGE of 32P-labeled P-adrenergic receptors partially purified from control and isoproterenol-desensitized turkey erythrocytes. Turkey erythrocytes were preincubated with [32P]orthophosphate for 20 h; 32P-labeled cells were then incubated for 4 h in buffer alone (lane 3), with 1 pi4 isoproterenol (lane 4),or with 1 pi4 isoproterenol plus 10 pi4 propranolol (lane 5 ) . P-Receptors were partially purified from membranes prepared from these cells by solubilization and affinity chromatography. Included in this panel for comparison are P-receptors labeled with [1251]pABCin membranes prepared from control (lane I ) and isoproterenol-desensitized (lane 2) erythrocytes from a separate experiment run on the same gel.

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toaffinity probe) labeled receptor from the membranes of control and desensitized cells. Lanes 3, 4 and 5 show 32P-labeledreceptor from control, desensitized, and agonist plus antagonist preincubated cells. This experiment demonstrates that the turkey erythrocyte P-adrenergic receptor is a phosphoprotein and that following desensitization the phosphate content of the receptor is increased two to three times compared to control. The ability of antagonist to block both the altered receptor mobility and increased 32P incorporation correlates well with the ability of the antagonist to prevent agonist-induced desensitization. This study demonstrates that the P-adrenergic receptor is specifically phosphorylated in turkey erythrocytes in response to prolonged challenge by agonist. Further evidence that desensitization of turkey erythrocyte adenylate cyclase is correlated with phosphorylation of the P-adrenergic receptor was provided by the work of Sibley et ul. (9). They found that under basal conditions the receptor contains 0.75 mol phosphate/mol while under maximally desensitized conditions the ratio increases to 2.34 mol/mol. They also demonstrated that the agonist dose-response curve as well as the time courses for desensitization and resensitization were identical for adenylate cyclase desensitization and receptor phosphorylation. The finding that incubation of turkey erythrocytes with cAMP and cAMP analogs only partially mimics isoproterenol in promoting adenylate cyclase desensitization and receptor phosphorylation suggests that only part of this process is mediated by the CAMP-dependent protein kinase. To directly assess the functionality of the P-adrenergic receptor from desensitized turkey erythrocytes, studies using receptor purified from both control and desensitized cells were performed (10). The purified receptors were implanted into phospholipid vesicles which were subsequently fused with Xenopus luevis erythrocytes. These cells contain N, and adenylate cyclase but little or no padrenergic receptor. As shown in Fig. 2, the fusion of reconstituted P-adrenergic receptor prepared from control cells establishes a sevenfold isoproterenol-induced stimulation of adenvlate cyclase activity in the previously unresponsive X. luevis erythrocytes. By contrast, receptor prepared from desensitized turkey erythrocytes establishes only a fourfold stimulation of adenylate cyclase. This represents a 45% decrease in the response and correlates very well with the extent of isoproterenol-induceddesensitization of the adenylate cyclase observed in the original crude membranes. These results thus demonstrate a direct relation between a stable modification of the P-adrenergic receptor due to hormoneinduced desensitization and a functional impairment of the receptor in a reconstituted system. Several studies have demonstrated that incubation of phorbol esters with duck and turkey erythrocytes leads to both desensitization of adenylate cyclase and phosphorylation of the P-adrenergic receptor (11, 12). That phorbol esters lead to desensitization of adenylate cyclase also had previously been shown in C6

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X&X

X&C

X&D

FIG.2. Fusion of X . luevis erythrocytes with affinity chromatography-purified and reconstituted P-adrenergic receptors from control and desensitized turkey erythrocytes. Receptor preparations were reconstituted into phospholipid vesicles and fused with X . laevis erythrocytes as described in (10). The same number of control and desensitized receptors were used in the reconstituion protocol and the same efficiency of insertion (20 to 25%) was observed in both cases. Abbreviations: B, basal: Iso, 50 )LM (-) isoproterenol; Is0 + Pro, 50 )LM (-)isoproterenol plus 50 pA4 (+)propranolol; PGE], 3 )LM prostaglandin El; and NaF, 10 mM NaF.

glioma (13) and mouse skin and epidermis (14). Since phorbol esters appear to act by stimulating the enzyme protein kinase C [reviewed in Ref. (IS)],these studies suggest that protein kinase C may be able to directly modulate cAMP levels via phosphorylation and desensitization of the adenylate cyclase-coupled 6-adrenergic receptor. In an attempt to more directly determine what protein kinases are involved in receptor phosphorylation, a cell-free system for desensitization of adenylate cyclase was developed (16, 17). Desensitization of adenylate cyclase in isolated turkey erythrocyte membranes was shown to require the presence of ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. In the cell-free system, isoproterenol promoted a 40-60% desensitization while cAMP led to a 20-30% decrease in adenylate cyclase activity. When the soluble fraction of the cell was not included, no desensitization of adenylate cyclase was observed. However, when isolated turkey erythrocyte membranes were incubated with purified CAMP-dependent protein kinase an 20-30% desensitization was observed. This effect is completely abolished by the specific inhibitor of the kinase. In contrast, only about one-half of the 40-60% desensitization induced in a turkey erythrocyte lysate by isoproterenol is blocked by the inhibitor of the CAMP-dependent protein kinase. Similarly only about one-half of the phosphorylation of the P-adrenergic receptor induced in the lysate system by isoproterenol is blocked by the specific CAMP-dependent protein kinase inhibitor. In this cell-free lysate system phorbol esters also promoted P-adrenergic receptor desensitization and phosphorylation ( I 7). This desensitization could be

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mimicked by incubating isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol esters. Calmodulin also promoted receptor phosphorylation and desensitization but to a much lesser extent than either isoproterenol or phorbol esters. The effect of calmodulin was entirely blocked by EGTA. These findings with the cell-free system suggest that (a) multiple protein kinase systems are capable of regulating P-adrenergic receptor function via phosphorylation reactions, and (b) CAMP may not be the sole mediator of isoproterenol-induced heterologous desensitization in these cells. We have directly studied the ability of several different protein kinases to phosphorylate the purified P-adrenergic receptor (18). In these studies, purified hamster lung P-adrenergic receptor was incubated with [ Y ~ ~ P I A TMg2+, P, and several different protein kinases. It was found that cGMP protein kinase. myosin light chain kinase, casein kinases I and 11, and rhodopsin kinase were unable to phosphorylate the receptor. Conversely, CAMP-dependent protein kinase was

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I INCUBATION TIME (HR)

2

FIG.3. Effect of isoproterenol on the time course of CAMP-dependent protein kinase-catalyzed padrenergic receptor phosphorylation. Purified hamster lung p-adrenergic receptor (0.14 @) was incubated with the catalytic subunit of CAMP-dependent protein kinase (0.34 @) in the presence or absence of 20 @ (-)isoproterenol at 25°C for the indicated period of time. Reactions were stopped by the addition of SDS sample buffer followed by electrophoresis on a 10%SDS-polyacrylamidegel. After drying and autoradiography, the gel was cut and counted to determine the stoichiometry.

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able to phosphorylate the receptor in a stoichiometric fashion. The time course of receptor phosphorylation by the CAMP-dependent protein kinase is shown in Fig. 3. It is seen that inclusion of the P-agonist, isoproterenol, promotes a 2- to 3-fold increase in the rate of receptor phosphorylation. This effect can be blocked by Pantagonists and suggests that the agonist induces a conformational change in the receptor which exposes the phosphorylation site(s). The stoichiometry of 0.5 mol 32P/mol receptor seen in Fig. 3 was found to be very dependent on the incubation conditions. When the receptor used was initially reconstituted in phospholipid vesicles a stoichiometry of 2 mol/mol could be attained. Highperformance liquid chromatography (HPLC) tryptic mapping of 32P-labeled receptor revealed two major phosphorylation sites both at serine residues. The phosphorylated receptor can be completely dephosphorylated by a high-molecular-weight phosphoprotein phosphatase. The rate of receptor dephosphorylation is also specifically enhanced 2- to 3-fold by isoproterenol again suggesting a conformational change in the receptor that exposes the phosphorylation sites. The functional significance of receptor phosphorylation was studied using ligand binding and reconstitution techniques. The P-adrenergic receptor, in addition to binding specific ligands, is able to interact with N, in a manner that promotes N, activation of adenylate cyclase. The interaction of purified receptor and N, in phospholipid vesicles is promoted by agonists and may be monitored by measuring the GTPase activity of N,. Figure 4 demonstrates that phosphorylated P-adrenergic receptor has a diminished ability to interact with N, with no apparent change in ligand binding. When the receptor is phosphorylated an 25% decrease in isoproterenol-promoted GTPase activity is seen relative to control receptor. The concentrations of isoproterenol which promoted 50% of the maximum GTPase activities were 136 and 125 nM for control and phosphorylated receptor, respectively. These studies provide a direct demonstration of regulation of the function of the isolated P-adrenergic receptor by CAMP-dependent protein kinase. Moreover, they suggest one possible general mechanism for heterologous regulation of adenylate cyclase-coupled receptors. Cyclic AMP generated in response to agonist stimulation of the enzyme activates the CAMP-dependent kinase. This kinase presumably phosphorylates multiple receptors, thus uncoupling them from productive interaction with N,. Although the structures of the various adenylate cyclase-coupled receptors are not known, it is reasonable to speculate that there are regions of strong homology since they all couple to the same N, molecules. Thus, all these receptors might serve as substrates for the CAMPdependent kinase. Moreover, the ability of an agonist to change the conformation of the receptor in such a way as to make it a better substrate for the kinase provides an additional mechanism for fine-tuning the regulation. Thus, in addition to phosphorylation and desensitization of a variety of receptors there might be even more profound alterations of the particular type of receptor that is

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0 CONTROL B A R

0 PHOSPHORYLATED B A R

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FIG.4. Isoproterenol-promoted GTPase activity in phospholipid vesicles containing N, and phosphorylated or control receptor. Phospholipid vesicles containing P-adrenergic receptor, N,, and the catalytic subunit of CAMP-dependent protein kinase were incubated with 30 mM sodium phosphate, 10 mM tris-HC1, pH 7.2, 100 mM NaCI, 5 mM MgC12, 5 mM p-nitrophenyl phosphate, 50 pM AppNHp, and 250 k g of soybean phosphatidylcholine. Phosphorylation incubations additionally contained 50 pM ATP whereas control samples contained no ATP. After incubating for 2 hr at 25°C the samples were assayed for GTPase activity as a function of the isoproterenol concentration.

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occupied by its specific agonist. Finally, it is worth stressing, as previously noted, that kinases other than the CAMP-dependent kinase may well participate in these phosphorylation events.

B . HOMOLOGOUSDESENSITIZATION The possible involvement of P-adrenergic receptor phosphorylation in homologous desensitization has come under investigation. As previously noted, this form of desensitization is more specific for one class of agonist, is not CAMPmediated, and involves receptor sequestration within a poorly defined internalized compartment of membrane. Some evidence suggests that once “sequestered,’’ the receptors may be functionally normal [Ref. (19), as assessed by reconstitution approaches]. However, other evidence suggests that, especially at very early time points in the desensitization process, a functional alteration in the receptors may occur (20). Sibley et al. (21) have documented that homologous desensitization of the padrenergic receptor in the frog erythrocyte is associated with stoichiometric

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phosphorylation of the receptor. Other agents that raise cAMP levels in the cells without desensitizing the P-adrenergic receptor, such as PGE, , do not lead to padrenergic receptor phosphorylation under these conditions. This is consistent with the apparent lack of dependence of homologous desensitization on cAMP generation. Whether the receptor phosphorylation occurring during homologous desensitization is involved in triggering receptor sequestration, in directly uncoupling the receptor, or both, remains to be determined. There is also no evidence bearing on the nature of the kinases involved, other than the presumption that it is not the CAMP-dependent protein kinase.

111. Rhodopsin The rod outer segment of the retina converts a light stimulus into a change in the intracellular concentration of cGMP [reviewed in Ref. (22)l.This process is mediated by the GTP-dependent interaction of rhodopsin, a receptor for light, and transducin, a guanine nucleotide regulatory protein which is structurally similar to N, and Ni (23). Activated transducin is able to directly stimulate the enzyme cGMP phosphodiesterase leading to a decrease in intracellular cGMP concentrations. A possible role of phosphorylation in the regulation of this system was noted by three independent groups in th.: early 1970s when they discovered a light-dependent phosphorylation of rhodopsin (24-26). The kinase involved in the phosphorylation appears to be !iighly specific for freshly bleached rhodopsin as a substrate (27-29). Under optimal conditions as many as 9 or 10 phosphate groups are incorporated into serine and threonine residues in the carboxyl-terminal region of rhodopsin (30, 31). Several groups have suggested that phosphorylation of rhodopsin is a mechanism to turn off phosphodiesterase activity (32-36). This was based on their findings that ATP suppresses light-stimulated activation of phosphodiesterase and GTP binding to rod membranes. The inability of phosphorylated rhodopsin to activate phosphodiesterase (via a reduced interaction with transducin) was directly demonstrated by Shichi er al. (37). In these studies, rhodopsin species containing either 0, 1, or 2 mol phosphate/mol rhodopsin were isolated by electrofocusing. The ability of these three rhodopsin species to activate transducin in phospholipid vesicles was then studied. Rhodopsin with 1 mol phosphate/mol was only 4% as active as control (no phosphate) whereas rhodopsin with 2 mol/mol was completely inactive. While the phosphorylation of rhodopsin inhibits its interaction with transducin (37), it also appears to enhance its interaction with a 48,000-dalton protein (38).This major soluble protein in rod cells appears to compete with transducin for binding to phosphorylated rhodopsin and thus may also be involved in regulating phosphodiesterase activity (38). These studies support the notion that light-activated phosphorylation of rhodop-

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sin directly leads to decreased activation of cGMP phosphodiesterase. This may represent a mechanism for light adaptation analogous to the desensitization of adenylate cyclase-coupled receptors discussed in Section 11.

IV. The Nicotinic Acetylcholine Receptor The nicotinic acetylcholine receptor (also see Chapter 11) is an integral membrane protein that functions as a ligand-gated ion channel at the vertebrate neuromuscular junction (39). The acetylcholine receptor has been most extensively studied in the Torpedo electric organ and exists as an oligomer of four polypeptide chains with masses of 40 (a),50 (p), 60 (y), and 65 ( 6 ) kilodaltons with a stoichiometry of a2 py6 (40).Phosphorylation of the acetylcholine receptor has been shown to occur both in vivo and in vitro with as rnany as 9 phosphoserines being identified, distributed 1 , 1, 2, and 5 among the a,p, y, and 6 subunits, respectively (41). Postsynaptic membranes rich in the acetylcholine receptor appear to contain both endogenous protein kinase (42-44) and protein phosphatase activities (45). The endogenous protein kinases include the CAMP-dependent protein kinase which phosphorylates the y and 6 subunits of the receptor on serine residues (46, 47). This phosphorylation can be completely blocked by the specific inhibitor of the CAMP-dependent protein kinase. Phosphorylation studies of purified acetylcholine receptor with the catalytic subunit of CAMPdependent protein kinase also demonstrate phosphorylation of the y and S subunits with stoichiometries of 1.O and 0.89 mol 32P/mol receptor, respectively (46). Endogenous protein kinase C can also phosphorylate the y and 6 subunits of the receptor on serine residues (48).In addition, an endogenous tyrosine protein kinase which phosphorylates the p, y, and S subunits of the receptor has been reported (49).This kinase phosphorylates a single tyrosine residue on each of the subunits with stoichiometries of 0.5 mol/mol attained in postsynaptic membranes. It has also been reported that purified pp60""' of Rous sarcoma virus and A431 cell membranes, which are rich in EGF receptor, both specifically phosphorylate purified acetylcholine receptor on the p, y, and 6 subunits (49).While the acetylcholine receptor can be extensively phosphorylated both in vitro and in vivo, the effects of phosphorylation on the function of the receptor are unknown.

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V. The Receptors for EGF and Insulin The EGF receptor mediates a wide array of biological effects including increased glycolytic activity, stimulation of amino acid and sugar transport, and increased rates of protein, RNA, and DNA synthesis (50) (also see Vol. XVII,

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Chap. 7). One of the earliest events after EGF binding is activation of a tyrosinespecific protein kinase which appears to be intrinsic to the EGF receptor (51-54) (see Vol. XVII, Chapter 6). Autophosphorylation of the EGF receptor occurs at one tyrosine residue in vivo (52, 55), whereas in vitro as many as four tyrosines are phosphorylated (55). In addition, extensive phosphorylation of the receptor at both serine and threonine residues occurs in vivo (52). Several lines of evidence suggest that protein kinase C is involved in the phosphorylation of the EGF receptor. It has been demonstrated that tetradecanoyl phorbol acetate (TPA) and other active tumor-promoting phorbol esters can modulate the binding of EGF to its receptor (56-65). Depending upon the cell type, phorbol esters appear to cause either a reduction in the number of EGF receptor sites (56-59) or a decrease in the affinity of the receptors for EGF (60-65). Phorbol ester treatment also stimulates phosphorylation of the EGF receptor at serine and threonine residues and appears to inhibit the tyrosine kinase activity of the receptor leading to reduced phosphotyrosine levels (64-67). Similar results are observed when diacylglycerol is used, again implicating protein kinase C (68). Protein kinase C can directly phosphorylate purified EGF receptor at three threonine residues and again results in decreased tyrosine kinase activity (66). Since EGF can enhance Ca2 influx and diacylglycerol formation (69), both endogenous activators of protein kinase C, this may represent a mechanism for feedback inhibition of the receptor tyrosine kinase. Phosphorylation of the EGF receptor by CAMP-dependent protein kinase has also been demonstrated in vitro (70, 71). However, a physiological role of EGF receptor phosphorylation by this kinase remains to be established. Insulin binding to its receptor also triggers a wide variety of biological effects (72). One of the earliest responses is phosphorylation of the P-subunit of the insulin receptor. Like the EGF receptor, the insulin receptor is phosphorylated on tyrosine by a receptor-associated tyrosine kinase (73-77) (see Vol. XVII, Chapter 7). The receptor is also extensively phosphorylated on serine and threonine residues. Several studies suggest that protein kinase C is involved in insulin receptor phosphorylation. Phorbol esters appear to decrease the affinity of insulin for its receptor in several cell lines (78, 79) although in some cells no effect on insulin binding was observed (57, 60. 80). Phorbol esters also promote phosphorylation of the insulin receptor (80, 81) and appear to inhibit insulin-induced receptor phosphorylation and insulin action (80). These studies suggest that phosphorylation of the insulin receptor regulates insulin action. +

VI. Other Membrane Receptors Mast cells and basophils have cell-surface receptors that bind IgE. The IgE receptor is composed of an a component of M, 45,000 and a P component of

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33,000. Phosphorylation studies with a rat basophilic leukemia cell line have demonstrated a non-IgE-mediated phosphorylation of the p component of the receptor (82). In contrast to these findings Hempstead et al. (83, 84) have shown phosphorylation of both the a and p components of the receptor in rat mast cells. Additionally, when these cells were stimulated with antigen a rapid increase (55% in 15 s) in the phosphorylation of the a component was observed with no apparent change in p component phosphorylation. These studies suggest that increased phosphorylation of the a component of the receptor may be part of the antigen-stimulated process leading to mediator secretion. The transferrin receptor is a 180-kDa phosphorylated glycoprotein consisting of two identical subunits. This receptor mediates endocytosis of transferrin resulting in cellular uptake of Fe2+. The signal for receptor-mediated endocytosis, other than ligand binding, is unknown. Two groups have shown, however, that phorbol esters induce a 10- to 20-fold increase in phosphorylation of the transferrin receptor as well as an 50% decrease in the number of cell-surface transferrin receptors (85, 86). While it is tempting to speculate that phosphorylation may be the signal for receptor-mediated endocytosis this does not appear to be the case in HL60 cells (85).In these cells, transferrin does not induce phosphorylation of the receptor nor inhibit phorbol ester-induced phosphorylation of the transfenin receptor. However, phorbol ester-induced phosphorylation of the transferrin receptor may well be acting as a signal for receptor internalization. M,

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14. 15.

Lefkowitz, R. J., Stadel, J. M., and Caron, M. G. (1983). Annu. Rev. Biochem. 52, 159. Stiles, G. L., Caron, M. G., and Lefkowitz, R. J . (1984). Physiol. Rev. 64, 661. Harden, T. K. (1983). Pharmacol. Rev. 35, 5. Sibley, D. R., and Lefkowitz, R. J . (1985). Nature (London) 317, 124. Stadel, J . M., Nambi, P., Lavin, T. N., Heald, S. L., Caron, M. G., and Lefkowitz, R. J. (1982). JBC 257, 9242. Zallor, M. J . , Kerlavage, A. R.,and Taylor, S. S. (1979). JBC 254, 2408. Shih, T. Y., Weeks, M. O., Young, H. A., and Scolnick, E. M. (1979). Virology 96, 64. Stadel, J . M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., and Lefkowitz, R. J. (1983). PNAS 80, 3173. Sibley, D. R., Peters, J. R., Nambi, P., Caron, M. G., and Letkowitz, R. J. (1984). JBC 259, 9742. Strulovici, B., Cerione, R. A., Kilpatrick, B. F., Caron, M. G., and Lefkowitz, R. J. (1984). Science 225, 837. Sibley, D. R., Nambi, P., Peters, J . R., and Lefkowitz, R. J . (1984). BBRC 121, 973. Kelleher, D. I . , Pessin, J . E., Ruoho, A. E., and Johnson, G. L. (1984). PNAS 81, 4316. Mallorga, P., Tallman, J. F., Hennebury, R. C., Hirata, F., Strittmatter, W. T., and Axelrod, J. (1980). PNAS 77, 1341. Belman, S., and Carte, S. J. (1980). Nature (London) 284, 171. Nishizuka, Y. (1984). Narure (London) 308, 693.

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