Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

Studies on the nature of interaction between follicle-stimulating hormone and the testicular receptors

Two previous reports from this laboratory showed that the binding of 125I-labeled human choriogonadotropin and 125I-labeled human luteinizing hormone to rat testicular receptors is partially irreversible and the binding parameters obtained from Scatchard analysis of the data can be drastically altered simply by changing the reaction volume of the binding assays (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim, Biophys. Acta 584, 407--435; Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim, Biophys. Acta 584, 436--453). It is reported herein that the binding reaction between follicle-stimulating hormone and testicular receptors displays very similar characteristics. The results support the previous conclusion that receptor concentrations fluctuate in the membranes and that the extent of their loss from tissue membranes in vitro is dependent upon time and temperature of incubation, the volume of buffer present, and the quantity of hormone used.     

Gonadal receptors. I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone.

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (Kd) and number of hormone binding sites (Bmax) are highly sensitive to changes in hormone and/or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher Kd as well as Bmax values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436--453).     

Effect of season and photoperiod on the follicle-stimulating hormone receptors in a subtropical bird

Annual changes in and photoperiodic influence oh the weight of gonads, a parameter of gonadal activity, are much smaller in female birds than in males. Effect of season and photoperiod on the follicle-stimulating hormone receptors in the testis or ovary was studied using a subtropical weaver finch. The number of follicle-stimulating hormone binding sites per unit testicular weight showed a peak in the non-breeding phase; while the total number of binding sites per two testes was maximal in the breeding phase and minimal in the regressive phase. In contrast, seasonal changes in follicle-stimulating hormone binding sites in the ovary were less marked. Exposure to short-day during the breeding phase induced marked decreases in the numbers of binding sites per unit testicular weight and per two testes. These numbers markedly increased after transfer to long-day during the non-breeding phase. However, there was no significant effect of short-day or long-day exposure on follicle-stimulating hormone binding sites in the ovary. These results suggest that photoperiod is an effective environmental factor in the regulation of follicle-stimulating hormone receptors in the testis and the effect is manifested by pronounced changes in the testicular weight during annual breeding cycle.     

Chimeric GnRH-LH receptors and LH receptors lacking C-terminus palmitoylation sites do not localize to plasma membrane rafts

Luteinizing hormone and gonadotropin releasing hormone receptors (LHR and GnRHR, respectively) are G protein-coupled receptors with important functions in reproduction. We have developed chimeric GnRHR-LHR that contain the full GnRHR coupled to various forms of the LH receptor C-terminus to explore the role of the LH receptor C-terminus in raft localization of the receptor and signaling. Addition of the full-length LHR C-terminus to GnRHR resulted in localization of the resting chimeric receptor in the bulk membrane rather than plasma membrane rafts as has been reported for the wild-type GnRHR [A. Navratil, S. Bliss, K. Berghorn, J. Haughian, T. Farmerie, J. Graham, C. Clay, M. Roberson, Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK, J. Biol. Chem. 278 (2003) 31593-31602]. With truncation of the LHR C-terminus, approximately 3% of chimeric receptors appeared in low density membrane fractions. Palmitoylation of sites on the LHR C-terminus appears important for raft localization. Mutations to C-terminus palmitoylation sites eliminated translocation of LH receptors from the bulk membrane to rafts upon binding of hCG although these mutant receptors retained the ability to signal via cAMP.     

Identification of functional receptors for vasoactive intestinal peptide and neurotensin in the human submandibular gland duct cell line, HSG-PA.

The HSG-PA human submandibular gland adenocarcinoma cell line has attracted attention recently as a potentially useful cell culture model for studies of salivary duct cell function and regulation. These cells possess a variety of morphological and biochemical markers found in salivary duct cells. Recently, muscarinic cholinergic receptors coupled to inositol intracellular Ca2+ mobilization (He et al., Eur. J. Physiol., 413 (1989) 505-510) and K+ fluxes (Ship et al., Am. J. Physiol., 259 (1990) C340-C348) have been identified in HSG-PA cells. In this study, we report the presence in these cells of functional receptors for two neuropeptides, vasoactive intestinal peptide (VIP) and neurotensin. Receptors for both peptides were labeled in intact cell radioligand binding studies and exhibited pharmacological profiles similar to receptors found in other tissues. There was close agreement between binding Ki values and the ED50 values for stimulation of second messenger production and modulation of K+ efflux, with all values between 1 and 5 nM. Whereas neurotensin stimulated K+ efflux dramatically, VIP alone had no effect but enhanced the response to neurotensin. These studies thus represent the initial documentation of functional receptors for VIP and neurotensin in a cell line of salivary duct cell origin.     

Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies

A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors [Sullivan, W. P., Beito, T. G., Proper, J., Krco, C. J., & Toft, D. O. (1986) Endocrinology (Baltimore) 119, 1549-1557] cross-reacts with the Mr 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with [3H]promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. A second purification step by diethylaminoethyl chromatography gives further enrichment to 3720 pmol/mg of protein (or 44% purity) to yield essentially two proteins, 120-kilodalton (kDa) B receptors and a 76-kDa non-steroid binding protein, each in approximately equivalent amounts. B receptors purified under these conditions are transformed and biologically active. They were maintained as undegraded 120-kDa doublets and retained both hormone and DNA binding activities. Isolated B receptors were free of the 90-kDa non-steroid binding protein observed to be associated with 8S untransformed receptors in other systems and were free also of the non-hormone binding 105-108-kDa B antigen described previously to copurify with chick PR. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG1), B-64 (IgG1), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG1), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology. These new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors.     

Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.     

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors.

The crystal structures of complexes of human growth hormone (hGH) with the growth hormone and prolactin receptors (hGHR and hPRLR, respectively), together with the mutational data available for these systems, suggest that an extraordinary combination of conformational adaptability, together with finely tuned specificity, governs the molecular recognition processes operative in these systems. On the one hand, in the active 1:2 ligand-receptor complexes, 2 copies of the same receptor use the identical set of binding determinants to recognize topographically different surfaces on the hormone. On the other hand, comparing the 1:1 hGH-hGHR and hGH-hPRLR complexes, 2 distinct receptors use this same set of binding determinants to interact with the identical binding site on the ligand, even though few residues among the binding determinants are conserved. The structural evidence demonstrates that this versatility is accomplished by local conformational flexibility of the binding loops, allowing adaptation to different binding environments, together with rigid-body movements of the receptor domains, necessary for the creation of specific interactions with the same binding site.     

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.     

Agonist-induced changes in beta-adrenergic receptors on intact cells

Competition by beta-adrenergic agonists and antagonists for 125I-pindolol binding sites on intact cells (1321N1 human astrocytoma and C62B rat glioma) was measured using short time binding assays as previously described (Toews, M. L., Harden, T. K., and Perkins, J. P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3553-3557). Preincubation of cells with agonists converted about half of the cellular beta-adrenergic receptors from a form exhibiting high affinity for the agonists isoproterenol and epinephrine and the antagonist sotalol to a form exhibiting much lower apparent affinity for these ligands in short time assays. Exposure to agonists did not alter the affinity of receptors for the antagonist metoprolol. This change in the ligand binding properties of the receptor was rapid (t1/2 = 1-2 min following a lag of about 0.5 min), reversible (t1/2 = 6-8 min), and dependent on the agonist concentration present during the preincubation (K0.5 = 15 nM for isoproterenol). Both isoproterenol and sotalol attained equilibrium with the high affinity receptors very rapidly but equilibrated only slowly with those receptors exhibiting low apparent affinity in short time assays. These results are interpreted in terms of a model which postulates that both the low apparent affinity in short time assays and the subsequent slow equilibration of hydrophilic ligands with these receptors result from agonist-induced internalization of a fraction of cell surface beta-adrenergic receptors. The relationship of this change in receptor binding properties to other aspects of agonist-induced desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system is discussed.     

Glucocorticoid receptors in human fibroblasts derived from normal dermis and keloid tissue

Hydrocortisone stimulates proliferation and System A amino acid transport in cultured human fibroblasts, while decreasing production of collagen. Fibroblasts isolated from keloid tissue have an unusual glucocorticoid response; they are hyporesponsive with regard to proliferation and collagen production but hyperresponsive with regard to the induction of System A amino acid transport (Russell, J. D., Russell, S. B., and Trupin, K. M. (1978) J. Cell. Physiol. 97, 221-229; Russell, S. B., Russell, J. D., and Trupin, J. S. (1982) J. Biol. Chem. 256, 9525-9531). We show here that despite these differences, the glucocorticoid receptors of keloid cells do not differ from those of normal dermal fibroblasts in steroid specificity, dissociation constant (Kd), total number of binding sites (Bmax), or nuclear binding of glucocorticoid-receptor complexes. A single glucocorticoid binding species of molecular weight 93,000 was found in both cell types. A monolayer assay for glucocorticoid receptor binding is described which facilitates analysis of multiple strains of cultured cells. This assay gives the same specificity and dissociation constants as the conventional cytosol assay, but it is more sensitive. The magnitude of induction of System A amino acid transport was found to be directly proportional to glucocorticoid receptor occupancy in both keloid-derived and normal fibroblasts. This induction requires serum, which can be replaced with 1 nM insulin.     

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.     

Coated vesicles from rat liver and calf brain contain cryptic mannose 6-phosphate receptors

Highly purified clathrin-coated vesicles, isolated from rat liver and calf brain, contain mannose 6-phosphate receptors. The coated vesicle receptors appear to have the same subunit molecular weight and similar binding affinity as the receptor previously purified from bovine liver and rat chondrosarcoma microsomes (Sahagian, G. G., Distler, J. J., and Jourdian, G. W. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4289-4293 and Steiner, A. W., and Rome, L. H. (1982) Arch. Biochem. Biophys. 214, 681-687). There is a considerable (greater than 60-fold) enrichment of receptors in liver coated vesicles as compared to liver microsomes. Experiments carried out with intact and detergent-disrupted coated vesicles indicated that the receptors face toward the inside of the coated vesicles. The data suggest that coated vesicles are involved in the intracellular transport of the mannose 6-phosphate receptor.     

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.     

Characterization of sulfonylurea receptors in isolated human pancreatic islets

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [³H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a K_d value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182–188, 1998. © 1998 Wiley-Liss, Inc.     

Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization

Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schr?der, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., H?llt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.