Hormonal desensitization: Comparison of the gonadotropin-hormone-releasing-hormone and β-Adrenergic receptor- effector systems

MOLECULAR

AND CELLULAR

NEUROSCIRNCES

3,91-105

(19%)

REVIEW Hormonal Desensitization: Comparison of the GonadotropinHormone-Releasing-Hormone and ,B-Adrenergic ReceptobEffector Systems THOMAS Departments

of Pediatrics

M. BADGER

and Physiology/Biophysics,

AND LAWRENCE

University

of Arkansas

Received for publication

E. CORNETT for Medical

December 16,

Sciences,

Little

Rock,

Arkansas

72205

1991

action, and much of what is currently known about desensitization of hormonal activation of adenylate cyclase The term desensitization refers to a cellular adaptation was first described in this model. The catecholamines that results in an attenuation of responsiveness to either norepinephrine and epinephrine, the native ligands for hormonal or pharmacological stimulation with time. Li/3-adrenergic receptors, have extremely profound effects gands such as peptides or catecholamines in the extraon smooth muscle, adipose tissues, myocardium, liver, cellular fluid bind to and interact with specific receptors brain, myometrium, formed elements in blood, and a located in the plasma membrane, activating a coupled number of hormone producing organs. Adrenergic agoeffector system that generates one or more intracellular nists and antagonists are used as pharmacologic agents signals (or second messengers) that produce the final ef- in the treatment of asthma, a disease in which desensifects of the hormone. Desensitization occurs when either tization may become an important factor in medical the receptor number is reduced or when receptor-mediated management. effector activation is impaired. Desensitization is believed These receptor systems were selected in this discussion to be an important biological process that provides both of the process of desensitization for several reasons. First, regulatory and protective mechanisms against possible each receptor is coupled to a different yet widely used nonphysiologic effects caused by hormones that are prosecond messenger system; phosphoinositide metabolism duced or secreted in abnormal amounts or patterns. Conand calcium for the GnRH receptor and cyclic AMP for sequently, understanding hormonal actions such as de- the /?-adrenergic receptor. Second, the ligands for these sensitization is essential when considering many forms receptor systems are structurally distinct and represent of medical intervention involving hormonal therapy. two completely different classes of hormones, each with Desensitization is a process that can develop in several unique properties that affect desensitization. Third, beendocrine systems. Two such systems have been described cause of differences in the experimental tools available in some detail in this review; the gonadotropin hormone for the study of each system, research has focused on releasing hormone (GnRH) receptor system and the @- different steps of hormone action to determine the mechadrenergic receptor system. Stimulation of secretion of anisms of desensitization. Thus, in many instances data luteinizing hormone (LH) from the anterior pituitary obtained from one system has provided insights into the gland by GnRH is a well studied ligand-receptor-effector mechanisms of the other system. system. Development of desensitization in this system will prevent successful hormonal therapy for ovulation II. DESENSITIZATION OF THE GNRH RECEPTOR induction or treatment of disorders such as idiopathic MEDIATED LH SECRETION hypothalamic hypogonadism. GnRH is a decapeptide and LH is a glycoprotein. The desensitization process involvGnRH is known to stimulate gonadotropin polypeptide biosynthesis, glycosylation, and secretion (l-3). GnRH ing the GnRH receptor is similar to that for other peptide hormone receptors thus far studied, as well as for glycois secreted episodically from the hypothalamus, resulting stimulation of the anterior pituitary and protein and protein hormone receptors, such as the in- in intermittent pulsatile secretion of LH. Episodic exposure of the pitusulin, thyroid hormone, and prolactin receptors. The adenylate cyclase coupled /3-adrenergic receptor is itary gland to GnRH is considered to be essential for maintenance of normal gonadotrope function (4). Studies one of the more extensively studied systems for hormone I. INTRODUCTION

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1044-7431/92 $3.00 Copyright 0 1992 by Academic Pram, Inc. All rights of reproduction in any form reserved.

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on GnRH action have demonstrated that pdsatile exposure to GnRH (i) maintains cellular LH pools (5); (ii) maintains GnRH receptor levels (4); and (iii) increases the mRNA levels for the two subunits of LH, LH-a, and LH-/3 (6). Thus, stimulation of gonadotropes by pulses of GnRH appears to regulate cellular mechanisms of the two most important processes necessary for maintaining LH stimulation of the gonads, i.e., LH synthesis and secretion. In vitro stimulation of perifused, dispersed anterior pituitary cells with hourly pulses of GnRH elicits pulsatile LH secretion much like that which occurs in uiuo (Fig. 1). Continuous infusion of the same hourly dose of GnRH results in an initial increase in LH secretion, followed by a decrease in LH secretion that returns toward basal levels, the hallmark of desensitization (Fig. 2). Stimulation of the desensitized cells with pulsatile GnRH at the concentrations used in Fig. 1 results in no further LH secretion. Thus, continuous infusion of GnRH induces a state of cellular refractoriness to further stimulation. On the other hand, as the concentration of GnRH pulses is increased, the desensitized cells respond to the same maximal level as the nondesensitized cells, but the dose-response curve is shifted to the right (Fig. 3). These relationships between exposure pattern and pituitary responsiveness have been used clinically to either increase

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FIG. 1. Effects of hourly pulses of LHRH (GnRH) on LH secretion. LHRH (20 pmol) was administered as a l-ml injection every hour to four columns and LH was measured in subsequent fractions. Reproduced by permission of the publisher, from Ref. (10).

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FIG. 2. Effects of continuous LHRH (GnRH) infusion on LH secretion. LH secretion is shown in response to a single bolus of 10 pmol LHRH (injected in 1 ml at the arrow) followed by an LHRH (10 n&f) infusion for 6 h. A and B are data from individual columns and C is the mean +_ SEM from four columns of the same experiment. Reproduced by permission of the publisher, from Ref. (10).

gonadotropin secretion in the case of idiopathic hypothalamic hypogonadism (7,8) or reduce gonadotropin secretion in the case of precocious puberty (9). The decreased hormonal response that occurs during desensitization could be due to either decreased sensitivity or decreased responsiveness as illustrated by Fig. 4. In the case of GnRH desensitization discussed above, desensitized gonadotropes were substantially less sensitive to GnRH. In addition, since the slope of the linear regression line for the GnRH dose response of desensitized cells was significantly less than the slope calculated from nondesensitized cells, less LH was secreted per increment increase in GnRH (10). Thus, GnRH-induced desensitization of the LH response in vitro appears to involve decreases in both sensitivity and responsiveness. Typically,

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3. LHRH (GnRH) dose response of desensitized cells. Disanterior pituitary cells were desensitized with 12 h of continuous Beginning approximately 6 h into the infusion, hourly pulses (75,125,250,500, or 1000 pmol) were administered as indicated arrows. A and B are data from individual columns and C is the SEM. Reproduced by permission of the publisher, from Ref. (10).

decreases in the sensitivity down-regulation.

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DESENSITIZATION

are associated with receptor

A. Receptor Desensitization Down-regulation is a term often used to identify that type of desensitization which results from decreases in the actual number of hormone receptor binding sites. Although it has been recognized for some time that the receptor concentration for a given hormone on the plasma membrane surface varies according to physiologic and pathologic states, the actual mechanisms regulating cell surface receptor numbers are not well understood. The apparent concentration or number of receptors can be altered by several general mechanisms, including: functional decreases in ligand binding without decreases in actual receptor number, removal of the receptor from the cell surface by translocation or internalization, changes in the synthesis or availability of receptors to ligand binding, changes in receptor degradation, prevention of receptor recycling, and masking or unmasking of extant receptors. Several GnRH analogs have been developed and serve as excellent experimental tools for studying cell surface receptors. Although direct experimental evidence for many other hormone receptors is lacking, GnRH receptors are considered to be evenly dispersed across the cell surface. Using bioactive GnRH analog derivatives (biotinylated or coupled with ferritin or rhodamine), GnRH re-

ceptors have been shown to be evenly distributed over the surface of gonadotropes (11-13). Binding of a ligand to a receptor can initiate or promote receptor/protein interaction and the receptor-ligand complex may aggregate. Although receptor aggregation has not been documented for all hormones, two distinct types of aggregation have been described; microaggregation and macroaggregation. Microaggregation occurs within milliseconds of ligand binding and is associated with hormonal actions (14). Macroaggregation, also called patching and capping, occurs minutes after hormone-receptor binding, well beyond the time required for hormonal signals to be transmitted across the membrane (15). Macroaggregation is followed by movement of the receptor-ligand complex into the cell (internalization of the receptor) (16, 17). Internalization reduces the number of receptors available for binding more hormone, thus resulting in an acute down-regulation. Internalization is an important step in the transport of the hormone to lysosomes for intracellular degradation in target cells (16, 17). Receptors with bound hormone face two possible fates upon internalization. Like the hormone bound to it, the receptor may undergo lysosomal degradation (18-20). However, in some hormonal systems a percentage of the internalized receptors return to the membrane (15, 21,22). This recycling event theoretically allows the receptor to be used over and over again. Those receptors that are degraded presumably must be replenished by de novo receptor synthesis if the cell is to maintain its level of responsiveness to a particular hormone. Recent reports indicate that a second class of receptors may be present in the plasma membrane, but are usually unavailable for ligand binding, and these have been

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FIG. 4. Types of resistance to hormone action. A dose-response curve that is shifted rightward indicates a decreased sensitivity, which is often cause by a receptor effect. A decrease in the maximal response suggests a decreased responsiveness and indicates a postreceptor event. Reproduced by permission of the publisher, from Ref. (128).

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termed “masked” receptors. In the case of GnRH, masked receptors become “unmasked,” and thus available for ligand binding, when the effector system is experimentally activated (23-25). The function of “masked” receptors and their relationship to the typical receptor is not well studied. It appears, however, that the unmasked GnRH receptors are selectively uncoupled from phosphoinositide metabolism (24, 25). Thus, the number of receptors on a target cell membrane can be altered by the rates of receptor synthesis, degradation, recycling, and/or unmasking. Any condition leading to decreases in ligand-receptor interaction, receptor synthesis or receptor recycling, or increased receptor degradation can lead to receptor downregulation and, therefore, result in lower hormonal responses at the same hormone dose, or desensitization. Results from several laboratories indicate that while GnRH-induced desensitization involves both GnRH receptor down-regulation and some decrease in pituitary LH concentration, other receptor events such as receptor modification or postreceptor events such as receptor-effector uncoupling must occur (26). Many hormones (e.g. insulin and thyroid-hormone-releasing hormone) have been reported to increase the rate of degradation of their own receptor (l&19,27,28), thus making the hormone itself an important regulator of hormonal receptor concentration on target cells. The term homologous down-regulation is used to refer to the process by which a hormone reduces the level of only its own receptor. When hormones negatively regulate the receptor level of other hormones, the term heterologous down-regulation has been used. The androgen-induced decrease in GnRH receptors on anterior pituitary cells (29) and the lower insulin receptor binding (30) observed with glucocorticoid treatment are examples of heterologous downregulation. Although desensitization in the form of receptor downregulation is an important regulatory mechanism of hormonal action, positive receptor regulation (receptor upregulation) is also important in establishing the responsiveness of a cell or tissue to a particular hormone. The term homologous receptor up-regulation is used to describe the condition in which a hormone can increase it’s own receptor on a target cell. For example, pulsatile luteinizing-hormone-releasing hormone (GnRH) can increase the GnRH receptor number on anterior pituitary cells (31,32) and prolactin will up-regulate prolactin receptors (33). It has been postulated that up-regulation of GnRH receptors is associated with increases in secretory granules that apparently contain either newly synthesized or recycled GnRH receptors (34). Heterologous up-regulation occurs when a hormone increases the numbers of receptor for other hormones. The FSH stimulated increase in LH receptor numbers (35) and the estrogen-induced increase in oxytocin receptor number in the uterus (36) are examples of heterologous receptor up-regulation.

B. Post-Receptor Desensitization As discussed above, receptor down-regulation or upregulation occurs in several hormonal systems, resulting in desensitization or sensitization of the hormone-stimulated response. In addition, some experimental evidence suggests that events following hormone-receptor binding, such as receptor-effector coupling, synthesis and degradation of second messengers, and responsiveness of second messenger, may also play a role in desensitization. Typical peptide hormones initiate biologic effects by binding to their receptor and activating an effector system that produces one or more second messengers necessary to elicit a response. Some insights into the mechanisms of GnRH induced desensitization can be gleaned from studies of each step between GnRH binding to the receptor on the gonadotrope and the eventual secretion or synthesis of LH during desensitization. 1. Receptor. Since high doses of GnRH have been shown to decrease the number of GnRH receptors (31, 37, 38), it seems reasonable to assume that at least some of the GnRH-induced desensitization seen in Figs. 2 and 3 was due to GnRH receptor down-regulation. There are data to suggest that neither desensitization nor sensitization of the gonadotrope by GnRH can be totally explained by changes in the receptor number (1, 2,31,39). For example, the desensitized state continues even after the GnRH receptor number on desensitized cells returns to control levels (31). However, desensitization appears to require both receptor occupancy by GnRH and activation of the second messenger system. Mere receptor occupancy by a ligand will not desensitize the system, as demonstrated by the inability of GnRH antagonists to produce desensitization (40). The requirement for both receptor occupancy and activation of the second messenger system is illustrated by results of innovative experiments from Conn’s laboratory in which an antagonist was converted to an agonist via conjugation of two antagonist molecules to an antibody, which in turn evoked both activation of the system and desensitization (41). In addition, the ability of high doses of GnRH to induce LH release in desensitized cells (Fig. 3) suggests that desensitization is mediated via the GnRH receptor. Photoaffinity labeling experiments revealed that no major structural differences could be detected between control and desensitized receptors (42). These results suggest that if ligand-induced desensitization occurs through modifications in the GnRH receptor, the changes probably involve more subtle effects such as conformational changes in the receptor. However, it has been observed that the a,-adrenergic receptor can be uncoupled from phosphoinositol metabolism by receptor phosphorylation (43). Whether this is the case with the GnRH receptor during desensitization is not known. 2. Receptor internalization. As mentioned above, internalization occurs too late to be involved in the LH

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release stimulated by a single pulse of GnRH, but it is possible that internalization could alter subsequent responsivity to GnRH-stimulated LH release. However, measurable desensitization occurred even when internalization was blocked (39). In addition, when the GnRH receptor number in desensitized cells returned to control levels, desensitization persisted (31), indicating that desensitization can occur independent of receptor downregulation. 3. G proteins. Far less is known about the possible involvement of guanine nucleotide-binding proteins (G proteins) in the actions of GnRH than is known for adrenergic systems. Evidence from Conn’s laboratory suggests that G proteins may couple GnRH receptors to inositol phospholipid hydrolysis and LH release. In the presence of ATP, guanine nucleotides stimulated accumulation of inositol phosphate and LH release (44). The blockade of these guanine nucleotide actions by a GnRH antagonist suggests that a G protein(s) is functionally associated with the GnRH recognition site. Desensitization could very well involve the phosphorylation of GnRH receptor associated ,G proteins or phospholipase C. Recent in vitro studies have demonstrated that sodium fluoride (NaF)-stimulated LH release is the same in control or GnRH-desensitized anterior pituitary cells (45). Based on these data, these authors speculate that (i) the site of NaF action on LH release is distal to the site of GnRH action for desensitization, (ii) GnRH-induced desensitization is caused by an action on the GnRH receptor or the mechanisms involving coupling of the receptor and phospholipase C-associated G proteins, and (iii) desensitization may be caused by phosphorylation of the GnRH receptor. 4. Phospholipase C, diacylglycerol, inositol phosphates, and protein kinase C. GnRH binding to gonadotropes has been linked to metabolism of phosphatidic acid and phosphatidylinositols (46, 47). Phospholipase C hydrolyses phosphatidylinositol 4,5-bisphosphate, forming inositol 1,4,5-trisphosphate (48). This product is apparently responsible for the release of intracellular Ca2+ stores from endoplasmic reticulum and in addition has been implicated in Ca2+ gating at the plasma membrane (49-52). Although a role for phospholipase C in desensitization has not been defined, it is possible that the longer term actions of inositol1,4,5-trisphosphate on Ca2+ may prove important in desensitization. The second immediate product of phosphatidyinositol4,5-bisphophate hydrolysis is diacylglycerol (DAG). DAG and Ca2’ apparently act as coactivators of protein kinase C. Phorbol esters have been found to activate protein kinase C (53) and to release LH (41, 54, 55), suggesting that at least part of the GnRH-induced LH release mechanism involves activation of protein kinase C. It was initially thought that inhibition of the DAG activation of protein kinase C might result in desensitization. However, cells

that have been depleted of protein kinase C can be desensitized and down-regulated by GnRH, suggesting that neither desensitization nor down-regulation requires protein kinase C activity (45,56,57). 5. Calcium mobilization. Maximal GnRH stimulated LH release requires extracellular Ca2+ (58). Omission or chelation of Ca2+ in extracellular medium, or pharmacological Ca2+ channel blockage, reduced depolarizationinduced or GnRH-induced LH releases in vitro from pituitary glands, and agents that increase intracellular Ca2+ release LH in a manner similar to that of GnRH (59-63). Ca2+ ionophore-induced LH release (a process that does not require GnRH activation of its receptor) does not exhibit desensitization, suggesting that GnRH-induced desensitization is not a Ca2+ mediated event. C. GnRH-Induced

Hormone Expression

GnRH-induced desensitization of the LH response to GnRH is related in part to the decrease in LH storage and the availability of LH for secretion. The lower pituitary LH content following chronic treatment with GnRH could be caused by several factors, including; reduced synthesis that does not keep pace with LH secretion and/ or degradation. For the most part, the literature suggests that expression of the rate-limiting LH subunit (LH-P) is decreased during desensitization, resulting in reduced LH synthesis. Long-term (days) in vivo exposure to GnRH or GnRH agonists results in decreased serum LH levels, decreased pituitary LH contents, decreased steady-state levels of LH-fl mRNA, and no changes in the steady-state levels of LH-(Y mRNA (64, 65). GnRH agonists also prevent the castration-induced rise in LH-/3 mRNA, but have no effects on the increase in LH-(U mRNA levels following castration. Long-term in vitro GnRH agonist exposure results in significant increases in free LH-a subunit and steady-state levels of LH-(Y mRNA (66). Together, these observations indicate that desensitization of the GnRHinduced LH response can be in part localized to LH synthesis. These data correlate well with clinical observations in GnRH-treated men and women, demonstrating severe decreases in circulating LH levels and elevated free LH(Y subunit (67, 68). A recent report suggests that the elevated free LH-(Y may be related also to diminished renal clearance (69). D. Summary Relatively little information is available on the mechanism of GnRH-induced desensitization of the LH response in the pituitary. It does seem clear that desensitization can be accounted for in part by receptor downregulation and that receptor internalization appears to be important in the down-regulation process. That portion of desensitization not associated with receptor loss requires GnRH binding and effector activation, but does not appear to require active protein kinase C. Ca2+ does

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not appear to mediate desensitization, thus effectively ruling out calmodulin and associated binding proteins in the mechanisms of desensitization. The areas not well studied include phosphorylation of the GnRH receptor, possible G proteins, or phospholipase C.

CORNETT

OUT

III. DESENSITIZATION OF &ADRENERGIC REcw-roRs While frequently referred to as a single receptor, particularly in the older literature, in mammals two @-adrenergic receptor subtypes, termed ,@iand &, have been characterized. The @-adrenergic receptor in the nonmammalian erythrocyte model systems that have been extensively used to study desensitization appear pharmacologically to be more closely related to the mammalian &adrenergic receptor than to the &-adrenergic receptor. The @-adrenergic receptor subtypes can be distin~ished pharmacologically and each shows a distinctive tissue ~stribution. Moreover, through the application of molecular cloning techniques, the mammalian &- and &adrenergic receptor subtypes have been shown to be products of separate genes (70, 71). However, the ,&adrenergic receptors share a common intracellular second messenger signaling pathway. Both receptor subtypes are linked in a stimulatory fashion to adenylate cyclase through G,, a guanine nucleotide regulatory protein (Fig. 5). Binding of agonists to the fll- and &adrenergic receptors leads to activation of adenylate cyclase and an increase in intracellular cyclic AMP concentrations. A detailed discussion of the coupling of @-adrenergic receptors to adenylate cyclase is beyond the scope of this overview. However, numerous review articles have been written on this subject (72-75). A. Plasma Membrane

Receptor Desensitization

Much of what is known currently about the phenomenon of desensitization as it applies to adenylate cyclasecoupled receptors was first observed with the P-adrenergic receptor. The first report of desensitization of catecholamine-stimulated adenylate cyclase activity was made using rabbit cerebellar slices (76). Initial treatment with norepinephrine followed by subsequent exposure to the hormone resulted in diminished cyclic AMP response. This effect appeared to be specific for catecholamines since previous exposure to norepinephrine had no effect on histamine-stimulated cyclic AMP generation in the same slices. The first insight into cellular mechanisms involved in desensitization of the &adrenergic receptor came with the demonstration of a loss of membrane-associated binding sites in erythrocytes isolated from frogs that had been previously injected with either norepinephrine or the @-adrenergic agonist isoproterenol (77). In a series of experiments using human astrocytoma cells and S49 lymphoma cell variants, desensitization of the

Mg+*ATP

Cyclic AMP

FIG. 6. Components of the p-adrenergic receptor-adenylate cyclase system. Binding of an agonist to the @-adrenergic receptor@-AR) results in stimulation of adenylate cyclase (AS.) activity and formation of cyclic AMP. A guanine nucleotide regulatory protein (G,) that is composed of three subunits, (Y, @ and y, couples the receptor to adenylate cyclase. @-adrenergic receptor was shown to be a multistep process (78). Upon exposure to agonists, the initial event was described as an “uncoupling” of the P-adrenergic receptor from adenylate cyclase. The extent of uncoupling was hormone concentration dependent and was reversible. Only with continual exposure to agonist was a loss of membrane-associated P-adrenergic receptors observed in radioligand assays. As was the case in the pioneering work of Kakiuchi and Rall (76), the @-adrenergic agonist-induced desensitization process in S49 lymphoma cells appeared to be relatively specific for the @-adrenergic receptor, since prostaglandin El-, NaF-, and guanylyl imid~phospha~-stimulated adenylate cyclase activities were not affected. Two separate lines of research have led investigators studying ,&adrenergic receptor desensitization to explore the possibility that one consequence of long-term exposure of cells to catecholamines might be receptor internulization or sequestration. First, direct observations suggested that desensitization of @-adrenergic receptor-mediated stimulation of adenylate cyclase activity was correlated with a loss of plasma membrane-associated /3-adrenergic receptors. Second, using immunocytochemical techniques a report indicated that peptide hormones such as prolactin (79) could be internalized, thus raising the possibility that the @-adrenergic receptor might undergo a similar process. @-Adrenergic receptor internalization was first demonstrated in frog erythrocytes (80). Following exposure of cells to isoproterenol, the appearance of specific agonist binding in the supernate fraction concomitant with decreases in particulate binding was observed. Significantly, the desensitized @-adrenergic receptors in frog erythrocytes exhibited agonist binding properties which were similar to those of receptors that were uncoupled from G, and adenylate cyclase (80, 81). That is, only the low affinity state of desensitized receptors was observed and guanine nucleotides had no apparent effect on agonist binding. Similarly, in human astrocytoma cells, binding

HORMONAL

DESENSITIZATION

of isoproterenol to desensitized P-adrenergic receptors in light vesicular fractions was shown not to be affected by the addition of guanine nucleotides (82). Since the effect of guanine nucleotides on the affinities of agonist binding to P-adrenergic receptors is mediated by G,, it was concluded that internalized fi-adrenergic receptors are “uncoupled” from G,. However, it was uncertain from these studies as to whether desensitized receptors were internalized either with or without G, and adenylate cyclase. A possible explanation for these observations was that desensitized /I-adrenergic receptors were “functionally,” but not physically, uncoupled from G, and adenylate cyclase. Desensitization might involve alterations in either the receptor or the G,, two conditions that would lead to decreased hormonal response. Experimental evidence which suggested that desensitized P-adrenergic receptors may not be functional simply because G, and adenylate cyclase were not co-internalized came from a study in which sequestered vesicular p-adrenergic receptors isolated from frog erythrocytes were fused to Xenopus Levis erythrocytes (83). fl-Adrenergic receptors are nearly undetectable in Xenopus erythrocytes by radioligand assays as is /3-adrenergic receptor-stimulated adenylate cyclase activity, but prostaglandin Elstimulated adenylate cyclase activity is observable. Thus, Xenopus erythrocytes express functional G, and adenylate cyclase. Using this reconstitution assay system, desensitized /3-adrenergic receptors were shown to retain their functionality as evidenced by their ability to mediate /Iagonist stimulated adenylate cyclase activity (83). Similarly, desensitized P-adrenergic receptors in light vesicular fractions of S49 lymphoma cells have been shown to stimulate adenylate cyclase in a reconstitution system (85). These results suggested that at least in the frog erythrocyte and S49 lymphoma cell models, the functional uncoupling of the /3-adrenergic receptor during desensitization was not due to modfications in the receptor itself that rendered it unable to couple to G,. Rather, the reduced agonist-stimulated adenylate cyclase activity following exposure to isoproterenol appeared to be a result of physical sequestration away from other effector proteins that are involved in cyclic AMP generation. Making use of one of the S49 lymphoma cell variants, cyc-, additional evidence in support of this possibility has been obtained in the frog erythrocyte system (81). The cyc- variant possesses adenylate cyclase but lacks functional G,, thereby making it unresponsive to P-agonists, guanine nucleotides and NaF (84). Desensitized fl-adrenergic receptors in sequestered vesicles were solubilized in cholate and the resulting extract was reconstituted with S49 lymphoma cell cyc- membranes (81). The results of these experiments demonstrated diminished ability to reconstitute NaF-stimulated adenylate cyclase activity in cyc- membranes, suggesting that neither G, nor the catalytic subunit is sequestered together into the light ve-

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sicular fraction with the ,&adrenergic receptor during desensitization. While the desensitization process involving the o-adrenergic receptor in the frog erythrocyte and S49 lymphoma cell results in an apparently otherwise functional receptor being sequestered away from the G,-adenylate cyclase complex, fl-adrenergic receptors appear to undergo both structural and functional alterations during the desensitization process in other cell types. For example, a structural alteration in the turkey fi-adrenergic receptor following desensitization has been noted as evidenced by decreased mobility of desensitized receptors on SDSpolyacrylamide gels (86). This decreased mobility on SDS-polyacrylamide gels upon long-term exposure to /3adrenergic agonists was shown to be a result of phosphorylation of the receptor, a form of receptor modification that is discussed in detail in a later section. It is of interest that by the use of similar methodologies, no differences in apparent molecular weights were noted between control and desensitized /3-adrenergic receptors isolated from frog erythrocytes (81). In studies with human astrocytoma cells, evidence has been provided which suggested that the early phase of /3adrenergic receptor desensitization could be separated into two distinct steps (87, 88). Following exposure to agonists, “uncoupling” or loss of hormonal responsiveness generally occurred before @-adrenergic receptors could be detected in light vesicular fractions. These data suggest that the functional alteration in the desensitized @-adrenergic receptor, as measured by decreased ability to stimulate adenylate cyclase in reconstitution assays, may occur very early in the desensitization process. Thus, the process of internalization may not be essential for either the ligand-receptor-mediated hormonal event or the process of desensitization. Moreover, it is possible that in some systems such as the frog erythrocyte and S49 lymphoma cell this functional receptor modification is reversed by the time the desensitized receptors are sequestered. In other cells, the functional alteration in desensitized ,&adrenergic receptors appears to reverse somewhat more slowly and, therefore, in these systems the internalized receptor is still unable to stimulate adenylate cyclase in reconstitution assays. B. Postreceptor Desensitization Thus far, consideration has been focused primarily upon alterations in receptor properties during the desensitization process. The results of studies that employed the S49 lymphoma cell cyc- variant (lacks G, and hence P-adrenergic-stimulated adenylate cyclase activity) were responsible for directing attention away from G, and adenylate cyclase as potential targets during the course of desensitization. Using the cyc- variant, agonist occupation of the P-adrenergic receptor in the absence of receptorG, coupling and subsequent cyclic AMP generation was shown nevertheless to result in desensitization (89, 90).

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The type of desensitization achieved in this experimental paradigm appeared to be the homologous form since in reconstitution assays with wild type membranes to assess receptor functionality, PGEi- and NaF-stimulated activities were only minimally affected (90). Thus, it would appear that with homologous desensitization, potential alterations in the membrane components involved in cyclic AMP generation in response to @-adrenergic agonist stimulation are limited to the fl-adrenergic receptor. This conclusion seems intuitively appropriate since functional alterations in shared effecters downstream from the receptor (e.g., G, and adenylate cyclase) would likely lead to diminished responsiveness to all hormones in a given cell that function through the same effector pathway, the hallmark of heterologous desensitization. Evidence for postreceptor alterations has been obtained in the case of heterologous desensitization. G, isolated from fibroblasts treated with PGE, appears to be less effective at restoring adenylate cyclase activity in cycmembranes compared to G, isolated from either untreated or isoproterenol-treated cells (91). Significantly, the form of desensitization observed with isoproterenol was the homologous type, which apparently involved “functional uncoupling” of the fl-adrenergic receptor from a fully active G,-adenylate cyclase complex. Similarly, treatment of avian erythrocytes with p-agonists appears to lead to heterologous desensitization associated with alterations in G, (92-94). For example, using reconstitution of solubilized G, into cyc- membranes as a measure of functionality, the activity of G, isolated from desensitized turkey erythrocytes is less than that of G, that was solubilized from untreated cells (95). A novel mechanism for heterologous desensitization has been suggested as the result of experiments with MadinDarby canine kidney (MDCK) cells (96). Glucagon treatment was found to lead to heterologous desensitization as judged by diminished glucagon-, PGEi-, and NaFstimulated adenylate cyclase activities. However, G, alterations were not associated with this form of desensitization. Rather, increases in the levels of the inhibitory guanine nucleotide regulatory protein, Gi, were noted. These findings suggest that a decrease in either the actual or functional ratio GJGi could be involved in the development of heterologous desensitization. C. Phosphorylation

of the @-Adrena@

Receptor

Biochemical and molecular techniques have been applied recently to the study of the mechanisms involved in desensitization of the /3-adrenergic receptor-adenylate cyclase system. To date, the primary focus has been placed upon agonist-induced @-adrenergic receptor phosphorylation as an initial step toward the desensitized state. Two separate lines of investigation led to the eventual direct demonstration of P-adrenergic receptor phosphorylation during the course of the desensitization process. The first piece of evidence came from an unlikely source,

CORNETT

when the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was shown to inhibit isoproterenol-stimulated cyclic AMP accumulation in mouse epidermis (97). Subsequently, additional evidence was presented which suggested that the inhibitory action of phorbol esters was a result of uncoupling of ,&adrenergic receptors from adenylate cyclase (98,99). The second line of evidence arose when it was shown that desensitization of the turkey erythrocyte ,&adrenergic receptor could be achieved by treating cells with a cell membrane-permeable cyclic AMP analog, dibutryl cyclic AMP (100). Since it is thought that the primary action of cyclic AMP is to activate protein kinases, the authors suggested that phosphorylation of the @-adrenergic receptor or other proteins may be involved in desensitization (100). The first direct evidence for ,&adrenergic receptor phosphorylation came with the demonstration that [3”P] incorporation into the /3-adrenergic receptor is higher in desensitized cells than in control cells and that purified ,f3-adrenergic receptor is a substrate for phosphorylation by purified catalytic subunit of cyclic AMP dependent protein kinase (86). Stoichiometry determinations have demonstrated that in the absence of agonists, approximately 0.7-1.0 mol phosphate is present per mole of padrenergic receptor. This ratio is increased to approximately 2-3 mol phosphate/mole receptor with maximal agonist-induced desensitization (101). Moreover, the stoichiometry of moles of phosphate incorporated was highly correlated with the degree of desensitization achieved at each dose of isoproterenol. Additional evidence in support of phosphorylation playing a role during the course of P-adrenergic receptor desensitization in the turkey erythrocyte system came with the establishment of a cell-free system in which the putative involvement of known protein kinases could be studied. In this system, it was shown that the extent of desensitization depended not only upon the concentration of @-adrenergic agonist but also the presence of ATP, Mgs+, and unidentified cytosolic factor(s) (102). In addition, desensitization and receptor phosphorylation could be induced by either phorbol esters or calmodulin (103). Moreover, isoproterenol-stimulated phosphorylation of the /3-adrenergic receptor could be attenuated by a cyclic AMP-dependent protein kinase inhibitor to the same extent as desensitization (103). Taken together, these results suggest that several different protein kinases, including cyclic AMP dependent protein kinase, protein kinase C, and Ca’+/calmodulin-dependent kinase; could phosphorylate the /3-adrenergic receptor, and therefore, cyclic AMP is probably not the only mediator of isoproterenol-induced desensitization in turkey erythrocytes. Heterologous desensitization of the avian erythrocyte /3-adrenergic receptor is associated with phosphorylation, an event that at least in part is mediated by cyclic AMP. In contrast, homologous desensitization of the /3-adrenergic receptor had been shown previously to occur in the

HORMONAL

99

DESENSITIZATION

absence of cyclic AMP generation (89,90). However, using frog erythrocytes, homologous desensitization of the fladrenergic receptor was shown to be correlated with receptor phosphorylation (104). Additional studies demonstrated that cyclic AMP-mediated phosphorylation of the mammalian fi-adrenergic receptor appears to occur by a process similar to that in avian erythrocytes. Purified hamster lung /3-adrenergic receptor is a substrate for cyclic AMP-dependent protein kinase in vitro and the phosphorylated receptor can be dephosphorylated by a purified phosphatase (105). Moreover, the P-adrenergic agonist isoproterenol enhances both phosphorylation and dephosphorylation. To date, the identities of the responsible protein kinases associated with homologous and heterologous desensitization have remained uncertain. However, it is evident that receptor phosphorylation could be the unifying mechanism that both forms of desensitization share in common. Since cyclic AMP-dependent protein kinase appears to be a likely candidate for mediating heterologous desensitization, the identification of the protein kinase(s) responsible for homologous desensitization received considerable attention. The S49 cell lymphoma cell line has played a key role in elucidating mechanisms involved in /3-adrenergic receptor desensitization by providing the essential initial information that led to the isolation of a novel protein kinase activity. Using the two mutants that are defective in the cyclic AMP-dependent pathway of P-adrenergic receptor action (cyc-, which is unable to synthesize cyclic AMP in response to /3-adrenergic stimulation, and kin-, which lacks cyclic AMP-dependent protein kinase activity) &adrenergic agonist-induced receptor phosphorylation was studied (106). The results of this study demonstrated that P-adrenergic stimulation could lead to receptor phosphorylation by a cyclic AMP-independent pathway. A kinase with specific @-adrenergic receptor phosphorylating activity was suggested, precedence for which existed, in the form of a rhodopsin kinase that serves to phosphorylate and desensitize rhodopsin, the visual pigment of the rod outer segment (107). Purification of a novel protein kinase activity from S49 kin- cells that preferentially phosphorylated agonist-occupied fi-adrenergic receptors has been reported and the enzyme has been given the name P-adrenergic receptor kinase or B-AR kinase (108). @-AR kinase activity is generally insensitive to known modulators of various protein kinases; for example, cyclic nucleotides, cyclic AMP-dependent protein kinase inhibitor, phorbol esters, and Ca2+/calmodulin. Moreover, B-AR kinase does not appear to phosphorylate common kinase substrates such as casein and histone. Interestingly, with increasing purification, phosphorylation activity of @-AR kinase toward hamster lung P2-adrenergic receptor decreases (109). However, activity can be restored with the addition of retinal arrestin (log), a protein that is involved in enhancing phosphorylation of rhodopsin by rhodopsin kinase and the sub-

sequent inhibition of the interaction between rhodopsin and transducin (110). By analogy, these results would suggest that a similar protein might be present in mammalian cells that express @-adrenergic receptor-stimulated adenylate cyclase activity. Recently, a complementary DNA encoding such a protein has been cloned and has been termed @-arrestin (111). The protein inhibits B-adrenergic receptor kinase-phosphorylated &adrenergic receptor function in a dose-related manner. A large percentage of P-AR kinase activity, normally associated with the cytosol, is translocated to the plasma membrane upon treatment of either S49 lymphoma cells or DDTi MF-2 smooth muscle cells with /3-adrenergic agonists (112). Additionally, prostaglandin El treatment is as effective as isoproterenol in promoting P-AR kinase translocation (112). Recent studies have now demonstrated that other membrane-associated receptors such as the cY2-adrenergic receptor (113) and the muscarinic cholinergic receptor (114) serve as substrates for phosphorylation by P-AR kinase. Therefore, it is possible that @-AR kinase may play a more general role as a functional regulator of receptors coupled to adenylate cyclase. However, this conclusion concerning the extent of the physiological actions of P-AR kinase remains uncertain with the recent purification and molecular cloning of a cDNA encoding P-AR kinase ( 115). The deduced 689 amino acid primary sequence contains a protein kinase catalytic domain with a relatively high level of sequence identity with similar domains in protein kinase C and cyclic AMP-dependent protein kinase. Results from Southern analysis of genomic DNA suggest that @-AR kinase may be a member of a multigene family of enzymes that phosphorylate hormone receptors (115). This finding leaves open the possibility that the preferred physiological substrate of P-AR kinase is the /3-adrenergic receptor. D. Molecular Mechanisms Desensitization

of P-Adrenergic Receptor

In this section, two aspects of the desensitization process are covered: the involvement of protein and/or RNA synthesis and recent attempts at the identification of specific sequences within the fl-adrenergic receptor primary amino acid sequence that might be involved in desensitization. In large part, the molecular tools required to explore the putative changes in /3-adrenergic receptor gene expression and post-translational modification in the receptor protein as possible mechanisms involved in desensitization have only recently been available. Early experiments used various inhibitors to investigate the possible involvement of protein and RNA synthesis in the development of the desensitized state. In general, from these studies it appeared that neither protein nor RNA synthesis was required for fl-adrenergic receptor desensitization. In initial experiments in which the protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin D were used, complete desensiti-

100

BADGERAND

zation was observed to occur following exposure to isoproterenol of either frog erythrocytes (116) or mouse Ehrlich ascites cells (117). While P-adrenergic receptor desensitization can apparently proceed in the absence of either RNA or protein synthesis in most systems, in rat glioma cells (118, 119) and cultured baby hamster kidney fibroblasts (120) inclusion of either cycloheximide or actinomycin D during exposure to /3-adrenergic agonists shortens the time required for the subsequent recovery from the desensitized state. These results suggest that an induced protein that turns over rapidly may be responsible for maintaining the desensitized state (119). To date, the identity of this protein is unknown, however, a potential candidate might be the hypothetical protein with properties similar to those of retinal arrestin, discussed in the previous section. With the molecular cloning of the &adrenergic (71) and &-adrenergic receptors (70), it is now possible to use recombinant DNA techniques to investigate molecular mechanisms of desensitization. Like other members of the adrenergic receptor family, based upon hydropathicity profiles of their primary amino acid sequences, the piand &adrenergic receptor subtypes share a common structural arrangement in the plasma membrane (Fig. 6). The central feature of this model is the assignment of seven stretches of relatively hydrophobic amino acids as membrane spanning domains, designated I through VII. With this arrangement, the amino terminus, which contains two potential N-linked glycosylation sites, is placed extracellularly and the carboxyl terminus is placed intracellularly. Of the six postulated connecting loops between membrane spanning domains, three each are proposed extracellularly and intracellularly. It is within the membrane spanning domains that the highest degree of homology between adrenergic receptor subtypes exists. Of particular importance to the understanding of desensitization, the carboxyl terminal tail is particularly rich in serine and threonine residues and consequently has received considerable attention as the site of receptor phosphorylation during desensitization. The relative importance of specific regions of the hamster &adrenergic receptor to the desensitization process has been analyzed by preparing a series of mutant receptors with deletions of varying lengths (121). Functional characterization of mutant receptors was determined following transfection and expression in mouse L cells that ordinarily do not express fi-adrenergic receptors. Treatment with isoproterenol of mouse L cells transfected with plasmids carrying wild-type @-adrenergic receptor displayed apparently normal receptor desensitization as measured by adenylate cyclase activity and receptor internalization (121). Interestingly, mutant receptors in which a significant portion of the carboxyl terminus was deleted, including consensus cyclic AMP-dependent protein kinase phosphorylation sites, displayed normal desensitization responses following agonist treatment (121).

CORNETT

Cl40 Cl40 YNH2

FIG. 6. Models of the presumed structures of the turkey erythrocytes P-adrenergic receptor and human &adrenergic receptor. (Upper panel) Turkey @-adrenergic receptor. The model is based on the primary amino acid sequence as deduced from a cloned cDNA (129). The receptor is composed of 433 amino acids. Seven hydrophobic domains consisting of 23-25 amino acids that are proposed to span the plasma membrane are labeled I through VII. The extracellular amino-terminal domain contains a consensus sequence for glycosylation (CHO). The carboxylterminal domain is located intracellularly. (Lower panel) Human /&adrenergic receptor. The model is based on the primary amino acid sequence as deduced from a cloned cDNA as reported by Kobilka et al. (130). The receptor is composed of 413 amino acids. Seven hydrophobic domains consisting of 24 amino acids that are proposed to span the plasma membrane are labeled I through VII. The extracellular aminoterminal domain contains two potential glycosylation sites (CHO). The third intracellular loop between membrane spanning domains V and VI and the carboxyl-terminal domain contain consensus cyclic AMP-dependent protein kinase phosphorylation sites. In addition, the carboxylterminal domain contains a serine- and threonine-rich region that may contain potential &adrenergic receptor kinase phosphorylation sites.

These findings have been essentially duplicated using mutant Pz-adrenergic receptors expressed in X. Levis oocytes (122). Either deletion of putative cyclic AMP-dependent protein kinase phosphorylation sites or removal of the serine- and threonine-rich carboxyl terminus had no effect on the extent of agonist-induced desensitization. The results of more recent experiments with additional receptor constructs suggests that with progressive truncation of the carboxyl terminus the relative rate of @-

HORMONAL

DESENSITIZATION

adrenergic receptor desensitization is slowed, but that the final outcome of the desensitization process is not affected appreciably (123, 124). A second line of research suggests that expression of the fl-adrenergic receptor gene may be reduced as part of desensitization. Incubation of DDTi MF-2 smooth muscle cells with fi-adrenergic agonists results in decreases in both fi-adrenergic receptor responsiveness and number (125). Moreover, relatively long-term (16 h) incubations with isoproterenol result in an up to 40% decrease in fiadrenergic receptor steady-state mRNA levels (125). The mechanism involved in the agonist-induced decline in /3adrenergic receptor mRNA levels appears to be posttranscriptional. /3-Adrenergic receptor mRNA half-life decreases from approximately 12 h in control cells to approximately 5 h in cells treated with agonists (126). Using S49 lymphoma cell mutants, @-adrenergic agonist-stimulated down-regulation of /3-adrenergic receptor mRNA levels appeared to require cyclic AMP-dependent protein kinase activity although elevated intracellular cyclic AMP was not necessary (127). Moreover, coupling between the /3-adrenergic receptor and G, was necessary in order to observe decreased mRNA levels. These results have led to the suggestion that at least two pathways exist by which cyclic AMP mediated desensitization of the &adrenergic receptor can proceed in Chinese hamster fibroblasts (123). The first pathway involves phosphorylation of the receptor, either by cyclic AMP-dependent protein kinase or by P-AR kinase, a step that from previous work discussed above would not appear to be obligatory for the full development of desensitization. The second pathway results in diminished steadystate levels of &-adrenergic receptor mRNA and therefore presumably decreased receptor expression. The molecular events leading to this condition have not been completely established. IV.

COMPARISON

OF DESENSITIZATION

GnRH AND ,&ADRENERGIC

OF THE

RECEPTOR SYSTEMS

From the preceding discussion, it is apparent that desensitization is an important process that plays a key role in determining a cell’s responsiveness to hormonal stimulation. Two major forms of desensitization, homologous and heterologous, have been characterized. Consequently, it is likely that multiple cellular mechanisms are involved in regulating the responsiveness of plasma membraneassociated hormone receptor systems. Both forms of desensitization appear to require receptor occupancy and activation of the associated second-messenger system. This was demonstrated persuasively by studies using GnRH antagonists. These antagonists bind to the receptor, but are not capable of activating the effector system nor of desensitization. Internalization, a process that sequesters receptors away from circulating hormone as well as effecters and

101

thereby reduces the number of functional receptors available for hormone binding, has been observed with homologous desensitization of both GnRH and /3-adrenergic receptors. This process, also termed down-regulation, may serve to regulate the level of hormonal stimulation of the target cell. Once internalized, the receptor ligand is apparently dissociated from the receptor. Little or no evidence has been reported for a specific intracellular role of GnRH or fl-adrenergic agonists. Although it appears that internalized GnRH is degraded by lysosomal action, the fate of internalized /3-adrenergic agonists is less well studied. Internalized receptors may, in some cases, be degraded or alternatively be returned to the plasma membrane (i.e. “recycled”). In contrast, heterologous desensitization of the P-adrenergic receptor does not result in receptor internalization. Rather, the desensitized receptor’s ability to interact with G, is functionally impaired, a state that could arise from covalent modification (e.g., phosphorylation) of either the receptor or G,. Phosphorylation of the P-adrenergic receptor has been associated with homologous desensitization as well. Molecular cloning and the subsequent determination of the primary amino acid sequences of the P-adrenergic receptor subtypes has allowed the identification of potential sites that when phosphorylated may either target the receptor for internalization or in the case of heterologous desensitization, functionally inactive the receptor. Agonist-occupied P-adrenergic receptor appears to be the preferred substrate for either cyclic AMP-dependent protein kinase or P-AR kinase. &Adrenergic receptor phosphorylation by cyclic AMP-dependent protein kinase appears to be associated with heterologous desensitization, while receptor phosphorylation by P-AR-kinase may initiate homologous desensitization. Phosphorylation of the GnRH receptor during the desensitization process has not been clearly established, but remains a possible mechanism for uncoupling the receptor from phosphatidylinositol-4,5bisphosphate hydrolysis. Postreceptor events of desensitization other than receptor phosphorylation are less well characterized. However, it appears that protein kinase C activity is not required for experimental desensitization by GnRH, and Ca2+ does not appear to mediate desensitization. Similarly, in P-adrenergic receptor systems, cyclic AMP generation does not appear to be required in order to observe desensitization. Finally, receptor gene expression may be reduced during desensitization. For example, the steady-state levels of mRNA encoding for the /3-adrenergic receptor are reduced with relatively long-term exposure to isoproterenol. The sequence of events leading to this state is not yet completely established, although cyclic AMP-dependent protein kinase activity seems to be necessary. Similar studies in the GnRH receptor system have not been reported. In summary, while desensitization is clearly an important process in hormone action, only a partial under-

102

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AND

standing of the cellular and molecular mechanisms involved has been achieved. Additional investigation of mechanisms of desensitization should ultimately help elucidate the complex regulation of receptor-coupled functions observed in a variety of cell types.

CORNETT is important 4209-4213.

15. Posner, peptide Recent

16.

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