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Impaired Desensitization of a Mutant Adrenocorticotropin Receptor Associated with Apparent Constitutive Activity

Department of Endocrinology (F.M.S., A.B., P.J.K., A.J.L.C.), Barts & the London, Queen Mary School of Medicine, London EC1A 7BE, United Kingdom; Department of Medicine (D.M.M., C.D.M.), University of Connecticut Health Center, Farmington, Connecticut 06030; Department of Internal Medicine (M.O.T.), University of Virginia Medical Center, Charlottesville, Virginia 22908; and Department of Physiology (L.H.), Semmelweis University Faculty of Medicine, Budapest H-1444, Hungary

Address all correspondence and requests for reprints to: A. J. L. Clark, Department of Endocrinology, Barts & the London, Queen Mary School of Medicine, West Smithfield, London EC1A 7BE, United Kingdom. E-mail: a.j.clark{at}mds.qmw.ac.uk.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
A naturally occurring ACTH receptor [melanocortin 2 receptor (MC2R)] mutation (F278C) has been identified in a subject with ACTH-independent Cushing’s syndrome. Functional characterization of this mutant receptor reveals that it is associated with elevated basal cAMP accumulation when compared with wild-type receptor-expressing cell lines. Dose responsiveness is similar between wild-type and mutant receptors in cell lines expressing similar numbers of binding sites. In view of the location of this mutation in the C-terminal tail of the MC2R, desensitization and internalization were investigated and found to be impaired. Inhibition of protein kinase A by H89 blocks wild-type MC2R desensitization and also results in increased basal activity, as does alanine substitution of Ser 280 in the C-terminal tail. Alanine substitution of Ser 208, the consensus protein kinase A phosphorylation target in the third cytoplasmic loop also results in a reduction in desensitization without significant change in basal activity or internalization. These findings suggest a novel mechanism is involved in the apparently constitutive activation of the MC2R in which failure of desensitization appears to be associated with enhanced basal receptor activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
CONSTITUTIVE ACTIVATION OF G protein-coupled receptors (GPCRs) as a result of single amino acid substitutions is a well described phenomenon that is associated with various endocrine and nonendocrine diseases. These include toxic thyroid adenoma (TSH receptor) (1), male pseudoprecocious puberty (LH receptor) (2), Jansen’s metaphysial chondrodysplasia (PTH/PTHrP receptor) (3), familial hypoparathyroidism (calcium-sensing receptor) (4), and congenital night blindness (rhodopsin) (5). Constitutive activation of the melanocortin 1 receptor (MC1R) is associated with coat color variations in several mammalian species (6).

It has been postulated that constitutive activation of the ACTH receptor (melanocortin 2 receptor, MC2R) might occur in adrenocortical pathologies associated with excessive glucocorticoid secretion and adrenal hyperplasia. ACTH is the primary physiological stimulus to glucocorticoid production, and pathological ACTH excess results in glucocorticoid oversecretion (Cushing’s syndrome) and adrenocortical hyperplasia. Inactivating mutations of the MC2R are accompanied by hypoplasia of the adrenal fasciculata and reticularis zones (7). Bilateral adrenocortical hyperplasia or isolated adrenal adenoma in the absence of ACTH excess are well recognized causes of Cushing’s syndrome. Inactivating mutations of the protein kinase A (PKA) type 1A regulatory subunit result in increased PKA activity and are associated with the Carney complex, which includes nodular adrenal hyperplasia and Cushing’s syndrome (8).

This reasoning has prompted the analysis of the MC2R in a range of adrenal hyperplastic and neoplastic pathologies. However, no missense or other mutations have yet been identified in two published studies (9, 10), suggesting that, at most, MC2R mutations are not a common cause of adrenal hyperplasia or tumor. However, there is evidence of overexpression of the MC2R mRNA in both aldosterone- and cortisol-secreting adenomas (11, 12), and MC2R gene loss of heterozygosity has been described in adrenocortical carcinoma (13).

Aloi et al. (14) previously reported a patient with episodic cortisol excess in the absence of detectable ACTH and bilateral adrenal hyperplasia, who was found to be homozygous for a germline missense mutation of the MC2R that resulted in the substitution of Phe 278 by Cys (F278C) in the C-terminal tail. We report here the functional characterization of this mutant receptor and demonstrate that it exhibits increased basal activity, which, we argue, is the consequence of a defect in receptor desensitization.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Functional studies of the MC2R have been significantly hampered by difficulties expressing this receptor in heterologous cells. However, the mouse adrenocortical Y6 cell line, or the closely related OS3 cell line, expresses no endogenous melanocortin receptor (15) and has been used successfully for MC2R expression (16, 17). Several stable Y6 cell lines incorporating either the empty pcDNA3 vector alone (cDNA3), pcDNA3 expressing the wild-type (WT) MC2R, or the mutant receptor (278C) were selected in G418. Three clonal cell lines of each type were used for further study.

Basal cAMP accumulation over a 60-min period is shown in Fig. 1. The mean basal cAMP accumulation for cDNA3, WT, and 278C cell lines was 342.0 (±60.9), 294.7 (±40.6), and 1229.0 (±273.9) pmol/mg protein, respectively (n = 4). The difference in cAMP accumulation between 278C and either empty vector or WT cell lines is statistically significant (P < 0.05; P < 0.005, respectively). There was no difference between the WT and cDNA3 cell lines.

View larger version (23K): [in this window] [in a new window]   Figure 1. Basal cAMP Production by Clonal Cell Lines Y6 clonal cell lines stably expressing the pcDNA3 vector alone (hatched bars), the WT MC2R (white bars), or the 278C mutant MC2R (black bars) were incubated for 60 min without stimulation in the presence of IBMX (n = 4). Accumulated cAMP (mean ± SEM) for three independent clones in each group is shown. Maximum ACTH binding was: vector only, all less than 0.05pmol/mg protein; WT MC2R, 1.3, 2.7, and 3.5 pmol/mg protein; 278C MC2R, 1.8, 7.0, and 9.9 pmol/mg protein (each measurement based on n = 3).

After stimulation by ACTH, cAMP accumulates in cells and the medium in the presence of a phosphodiesterase inhibitor. The rate of cAMP accumulation is shown in Fig. 2A and suggests a greater rate of cAMP accumulation by the 278C MC2R. Dose-response curves of WT and 278C cell lines to ACTH are shown in Fig. 2B. Y6 cells expressing pcDNA3 showed no response to ACTH. As before, the 278C-expressing cell line shows enhanced basal activity and a normal response to ACTH with an EC₅₀ value (4.31 ± 2.00 x 10^-9 M) that is not significantly different from that of the WT cells (9.30 ± 3.11 x 10^-9 M).

View larger version (19K): [in this window] [in a new window]   Figure 2. cAMP Responses of WT and 278C MC2R Expressed in Individual Y6 Clonal Cell Lines Expressing Either WT () or 278C () MC2R (Selected on the Basis of Similar Numbers of ACTH Binding Sites) A, Rate of cAMP accumulation after a single stimulus with ACTH[1–24] (10-8 M). Values are mean ± SEM (n = 3), expressed as a percentage of the maximal response in view of the differing basal and maximal values exhibited by the two receptor forms. B, Dose-response curves to ACTH[1–24] of the cell lines described in panel A (mean ± SEM; n = 6).

View larger version (31K): [in this window] [in a new window]   Figure 3. Pseudostructural Plot of the MC2R Showing Location of F278 in the C-Terminal Tail, the Location of the Two C-Terminal Serine Residues (filled circle) at Positions 280 and 294, and the Single Predicted PKA Phosphorylation Site at Serine 208 in the Third Cytoplasmic Loop

View larger version (18K): [in this window] [in a new window]   Figure 4. Desensitization and Internalization of F278C A, Desensitization of the ACTH (10-8 M) cAMP response of the WT () and 278C () MC2R-expressing cell lines after a preexposure to ACTH (10-8 M) for the times indicated. Results (mean ± SEM, n = 6) are expressed as a percentage of the response to a single stimulation of ACTH (10-8 M) in the presence of IBMX. *, P < 0.05; **, P < 0.005. B, Internalization of the WT () and 278C () MC2R-expressing cell lines after a single exposure to [125I]ACTH as determined using the acid wash technique. Values are mean ± SEM (n = 6) after subtraction of the nonspecific acid-insensitive extracellular counts. **, P < 0.005.

These findings imply that the F278C mutation results in increased basal activation and impaired desensitization and internalization. It seems highly likely that a single common mechanism may account for these three consequences. In the case of many GPCRs, desensitization results from phosphorylation of specific C-terminal or third cytoplasmic loop serine or threonine residues by G protein receptor kinases (GRKs) or second messenger-activated protein kinases, such as PKA or protein kinase C (20, 21). In the case of GRK phosphorylation, arrestin molecules bind to receptor preventing further G protein signal transduction and acting as adapter molecules to assist in clustering of the receptor in clathrin-coated pits and subsequent internalization, and in some instances, signaling along mitogenic pathways (22, 23, 24). Thus, defective arrestin binding could provide a common link between impaired desensitization and internalization. Studies with the ß₂-adrenergic receptor suggest that binding of arrestin is not required for desensitization of the receptor mediated by second messenger-activated protein kinases, although PKA-mediated phosphorylation has been implicated in internalization of the secretin receptor (25).

Conceivably, the location of codon 278 in the C-terminal tail of the MC2R might influence interaction with binding of arrestin or other molecules involved in the signal transduction process, or phosphorylation of one or both of the two serines located two and 16 residues downstream to the mutation. Neither of these serines forms a consensus site for phosphorylation by PKA. We have recently reported that in the case of the endogenous MC2R expressed by the mouse Y1 cell line, desensitization by ACTH is predominantly mediated by PKA (26), whereas internalization appears to be GRK dependent (27). Desensitization of the murine MC2R can be almost completely inhibited by the PKA inhibitor H89 or by mutagenesis of the single-consensus PKA phosphorylation site at Ser 208 in the third cytoplasmic loop (26). The mechanism of desensitization of the human MC2R has not been studied previously and, although the two receptors are 85% identical at the protein level, the possibility of a different mechanism in the human receptor cannot be excluded. Serines 208 (the consensus PKA site) and 280 are conserved in both species, although serine 294 is not present in the mouse.

In the presence of H89 the WT receptor showed marked loss of early desensitization similar to that found in the murine MC2R after this treatment (26) and, consequently, desensitization of the WT receptor in the presence of H89 was indistinguishable from that of 278C (Fig. 5). The 278C receptor showed no change in the pattern of desensitization after H89. It was notable in these studies that H89 was also associated with enhanced basal activity of the receptor [841.1 (±149.9) pmol cAMP/mg protein in the absence of H89; 2092 (±340.2) pmol/mg protein in the presence of H89; P < 0.01], once again supporting the notion of a link between failure of desensitization and apparent constitutive activity (Fig. 5B). Mutation of the consensus PKA phosphorylation site in the human receptor centered around S208 also resulted in impaired desensitization (Fig. 5C), although this impairment was apparently less marked, particularly at the 60-min time point, which did not differ significantly from the WT receptor. No significant increase in basal activity or any reduction in internalization was observed with the S208A receptor (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 5. PKA and MC2R Desensitization A, Desensitization of the cAMP response of the WT () and 278C () MC2R-expressing cell lines, and the WT MC2R-expressing cell line in the presence of 10-7 M H89 (). Values are mean ± SEM (n = 6). *, P < 0.05; **, P < 0.005 for comparison of WT and H89 points. B, Basal cAMP production of pcDNA3 vector alone (hatched bars), the WT MC2R (open bars), or the 278C mutant MC2R (solid bars) in the absence (-) and presence (+) of 10-7 M H89. Values are mean ± SEM (n = 6). C, Desensitization of WT () and S208A (PKA site) mutant MC2R (). Values are mean ± SEM (n = 3) *, P < 0.05; **, P < 0.005.

View larger version (21K): [in this window] [in a new window]   Figure 6. Role of C-Terminal Tail Serines 280 and 294 A, Basal cAMP production by individual WT receptor-expressing cell lines (open bars), 278C-expressing cell lines (hatched bars), and cell lines expressing the S280A (black bars) and S294A (black bars) mutant receptors. Values are means ± SEM (n = 4). Maximum ACTH binding was: WT MC2R, 19.4, 39.5, 19.7, and 30.1 pmol/mg protein; F278C MC2R, 17.2, 26.8, 10.1, and 8.7 pmol/mg protein; S280A MC2R, 7.3, 8.1, 15.1, and 10.4 pmol/mg protein; S294A MC2R, 24.2, 20.8, and 25.8 pmol/mg protein (each measurement based on n = 3). B, Desensitization of the cAMP response of WT cells (), S280A cells (), and S294A cells (). Values are means ± SEM (n = 3). *, P < 0.05 for comparison between S280A and WT points. C, Internalization of [125I]ACTH into WT cells (), S280A cells (), and S294A cells (). Values are means ± SEM (n = 3).

We propose that in the absence of agonist the MC2R, in common with the majority of GPCRs, exists predominantly in an inactive, nonsignaling state (R). A small proportion will exist in an active (R*) state that will generate a signal. This signal will activate desensitization mechanisms, which will serve to terminate this signal. In the case of the MC2R this seems to be an efficient process in that there is no significant difference in cAMP generation between vector-only and WT MC2R-expressing cells (Fig. 1). According to this model, any disturbance of receptor desensitization should result in enhanced basal signaling, as demonstrated by the 278C and 280A mutants.

This raises the question as to whether this is really a unique phenomenon likely to be limited to this mutation in this receptor or whether it may occur more generally without being recognized as the mechanism of activation. The observation that one other artificial mutation (S280A) and H89 can reproduce similar findings suggests that the latter option may be the case. Other events that impair desensitization, such as defective GRK or arrestin activity, may also result in constitutive activity of a number of receptor signal transduction systems. Indeed, congenital stationary night blindness may result from dominant mutations leading to constitutive activation of rhodopsin, or from homozygous defects of rhodopsin kinase or arrestin which lead to photobleaching of the retina (31). It is therefore conceivable that desensitization defects may contribute significantly to endocrine and other pathology including those that results from activation of mitogenic signal transduction systems as seen, for example, with G_s constitutive activation in acromegaly in pituitary and thyroid tumors.

Constitutively activating mutations of the MC2R have been sought in adrenal hyperplasia and tumors by a number of groups but have not been found (9, 10). The only genetic defect of this receptor in an adrenal tumor recorded to date is that of a hemizygous deletion of the MC2R gene in an adrenal carcinoma (13). An inevitable question is whether one would expect a constitutively activated MC2R to cause adrenocortical hyperplasia. It might be argued that the coexistence of this mutation and Cushing’s syndrome is a chance event. The literature surrounding the role of ACTH in adrenal growth is extensive and often conflicting. Recent evidence suggests that a locally cleaved N-terminal proopiomelanocortin-derived peptide cosecreted with ACTH may be the major adrenal growth factor (32), while other data suggests that, in some circumstances, ACTH can inhibit adrenal growth (33). Inactivation of the MC2R by mutation is associated with hypoplasia of the ACTH-responsive fasciculata and reticularis cell layers (7), while overexpression of ACTH by pituitary tumors consistently results in bilateral adrenocortical hyperplasia. We would argue therefore that the presence of this germline MC2R mutation is inextricably linked with the clinical phenotype in this patient.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
MC2R Expression Vectors and Stable Cell Line Generation
The full-length MC2R coding sequence was amplified by PCR from the index patient’s genomic DNA and subcloned ultimately into pcDNA3 (Invitrogen, San Diego, CA). WT human MC2R was similarly subcloned. Mouse Y6 cells (a gift from Professor Bernard Schimmer, University of Toronto, Toronto, Ontario, Canada) were grown DMEM:F10 (1:1 vol/vol) with 10% fetal calf serum, 10% horse serum, and penicillin/streptomycin. Cells were transfected with the appropriate pcDNA3 construction using calcium phosphate coprecipitation and selected in 200 µg/ml Genetecin (G418) (Life Sciences, Paisley, UK). Colonies were selected by ring cloning. The mutant receptors S208A, S280A, and S294A were generated using the QuikChange protocol (Stratagene, Amsterdam, The Netherlands), confirmed by DNA sequencing, and transfected and selected as above.

cAMP Accumulation
All studies were performed after preincubation of cells in serum-free medium. To assess basal cAMP accumulation, cells were exposed to 500 µl serum-free medium containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 60 min. Total cAMP (cellular and medium) was determined by a competitive protein binding assay (34). Protein estimation was performed using a Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay.

ACTH Stimulation
Cells were exposed to ACTH (10^-12 to 10^-6 M) at 37 C in the presence of IBMX (1 mM) before harvesting for cAMP and protein assay as described above. cAMP accumulation for time periods up to 30 min resulted in a comparatively variable response, and therefore 60 min was selected as the collection time for these studies. For desensitization studies, cells were exposed to 10^-8 M ACTH for 0–120 min as a prestimulation in the absence of IBMX, washed in serum-free medium, and reexposed to ACTH (10^-8 M) for 60 min in the presence of IBMX. Use of an acid wash step (10 min in 50 mM glycine, 100 mM NaCl, pH 3) between exposures to ACTH was found to make no difference to the desensitization kinetics observed. Preliminary experiments had shown that in the absence of IBMX detectable concentrations of cAMP do not accumulate in this cell line even after ACTH stimulation. For H89 experiments 10^-7 M H89 was added to the serum-free preincubation and the prestimulation and stimulation phases.

ACTH Competitive Binding Assay
The ACTH binding assay was performed on whole cells as described previously (17, 35) using [¹²⁵I]iodotyrosyl²³ ACTH(1–39) (Amersham Pharmacia Biotech, Little Chalfont, UK) in serum-free medium containing 0.5% BSA and 0.1% bacitracin. Mean total binding to WT cells was 3331 ± 387 cpm and mean nonspecific binding was 932 ± 286 cpm, indicating that approximately 28% of total counts were nonspecifically bound. Binding was determined using PRISM2 software (GraphPad Software, Inc., San Diego, CA) and applying nonlinear curve fitting to the homologous competition displacement curves. The method of the least squares was used to determine whether a one-site or two-site binding model gave the best fit.

Receptor Internalization Assay
Cells were seeded onto 12-well plates, and at 90% confluence transferred to serum-free medium for 1 h, then subjected to an acid wash (as described above) for 10 min, and then washed in serum-free media again, before exposure to 0.025 pmol [¹²⁵I]ACTH ( 900 cpm/well) in serum-free medium containing 0.5% BSA and 0.1% bacitracin, for 30 sec to 240 min, at 37 C. As described previously (19), cells were then transferred to ice and washed three times with 0.9% NaCl and then exposed to 500 µl ice-cold acid glycine (as described above) for 10 min. This was then carefully aspirated and counted on a -counter, representing the extracellular bound ACTH. Cells were then dissolved in 500 µl 0.5 M NaOH, 0.4% sodium deoxycholate, and the intracellular ACTH was determined by -counting. Internalization was expressed as the percentage intracellular/intracellular + acid-soluble ACTH. Nonspecific background counts amounted to 49.5 ± 0.7% (WT receptor) and 42.9 ± 4.6% (278C receptor) of total counts added and were subtracted from the actual counts measured in each experiment.

Statistical Analysis
All experiments were conducted in duplicate wells, and all results are the means of between three and six independent experiments as indicated. Values shown and plotted are the mean ± SEM. Statistical comparison was performed using nonpaired two-tailed Student’s t tests, and P values are indicated as *, P < 0.05; **, P < 0.005. Where no significance is indicated, P > 0.05.

	FOOTNOTES

 
This work was supported by a Wellcome Trust Clinical Training Fellowship Award (to F.M.S.) and a Wellcome Trust Collaborative Research Initiative Grant (to L.H. and A.J.L.C.). Additional support for F.M.S. and A.H.B. was received from the Joint Research Board of St. Bartholomew’s Hospital.

Abbreviations: GPCR, G protein-coupled receptor; GRK, G protein receptor kinase; IBMX, 3-isobutyl-1-methylxanthine; MC1R and MC2R, melanocortin 1 and -2 receptor, respectively; PKA, protein kinase A; WT, wild-type.

Received for publication March 11, 2002. Accepted for publication September 5, 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Parma J, Duprez L, Van Sande J, Cochaux P, Gervy C, Mockel J, Dumont M, Vassart G 1993 Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365:649–651[CrossRef][Medline]
2. Shenker A, Laue L, Kosugi S, Merendino JJ, Minegishi T, Cutler GB 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652–654[CrossRef][Medline]
3. Schipani E, Kruse K, Jüppner H 1995 A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268:98–100[Abstract/Free Full Text]
4. Baron J, Winer K, Yanovski J, Cunningham AW, Laue L, Zimmerman D, Cutler Jr GB 1996 Mutations in the Ca²⁺-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 5:601–606[Abstract/Free Full Text]
5. Rao VR, Cohen GB, Oprian DD 1994 Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature 367:639–642[CrossRef][Medline]
6. Robbins LS, Nadeau JH, Johnson KR, Kelly MA, Roselli-Rehfuss L, Baack E, Mountjoy KG, Cone RD 1993 Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72:827–834[CrossRef][Medline]
7. Clark AJL, Weber A 1998 ACTH insensitivity syndromes. Endocr Rev 19:828–843[Abstract/Free Full Text]
8. Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho-Chung YS, Stratakis CA 2000 Mutations of the gene encoding the protein kinase A type I- regulatory subunit in patients with the Carney complex. Nat Genet 26:89–92[CrossRef][Medline]
9. Light K, Jenkins PJ, Weber A, Perrett C, Grossman A, Pistorello M, Asa SL, Clayton RN, Clark AJ 1995 Are activating mutations of the adrenocorticotropin receptor involved in adrenal cortical neoplasia? Life Sci 56:1523–1527[CrossRef][Medline]
10. Latronico AC, Reincke M, Mendonca BB, Arai K, Mora P, Allolio B, Wajchenberg BL, Chrousos GP, Tsigos C 1995 No evidence for oncogenic mutations in the adrenocorticotropin receptor gene in human adrenal neoplasms. J Clin Endocrinol Metab 80:875–877[Abstract]
11. Reincke M, Beuschlein F, Lalli E, Arlt Y, Vay S, Sassone-Corsi P, Allolio B 1998 DAX-1 expression in human adrenocortical neoplasms: implications for steroidogenesis. J Clin Endocrinol Metab 83:2597–2600[Abstract/Free Full Text]
12. Arnaldi G, Mancini V, Constantini C, Giovagnetti M, Petrelli M, Masini A, Bertagna X, Mantero F 1998 ACTH receptor mRNA in human adrenocortical tumors: overexpression in aldosteronomas. Endocr Res 24:845–849[Medline]
13. Reincke M, Mora P, Beuschlein F, Arlt W, Chrousos GP, Allolio B 1997 Deletion of the adrenocorticotropin receptor gene in human adrenocortical tumors: implications for tumorigenesis. J Clin Endocrinol Metab 82:3054–3058[Abstract/Free Full Text]
14. Aloi JA, Carey RM, Thorner MO, Malchoff CD, Orth DN, Walsh B, Macgillivray D, Malchoff D, Episodic, ACTH-independent, Cushing’s syndrome associated with a point mutation of the ACTH receptor. Program of the 77^th Annual Meeting of The Endocrine Society, Washington DC, 1995, p 99 (Abstract OR39)
15. Schimmer BP, Kwan WK, Tsao J, Qiu R 1995 Adrenocorticotropin-resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor. J Cell Physiol 163:164–171[CrossRef][Medline]
16. Yang YK, Ollmann MM, Wilson BD, Dickinson C, Yamada T, Barsh GS, Gantz I 1997 Effects of recombinant agouti-signalling protein on melanocortin action. Mol Endocrinol 11:274–280[Abstract/Free Full Text]
17. Elias LLK, Weber A, Pullinger GD, Mirtella A, Clark AJL 1999 Functional characterization of naturally occurring mutations of the human adrenocorticotropin receptor: poor correlation of phenotype and genotype. J Clin Endocrinol Metab 84:2766–2770[Abstract/Free Full Text]
18. Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG, Lefkowitz RJ 1995 Phosphorylation and desensitization of the human ß-adrenergic receptor. J Biol Chem 270:17953–17961[Abstract/Free Full Text]
19. Hunyady L, Bor M, Balla T, Catt KJ 1994 Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of the AT₁ angiotensin receptor. J Biol Chem 269:31378–31382[Abstract/Free Full Text]
20. Ferguson SS, Barak LS, Zhang J, Caron MG 1996 G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74:1095–1110[CrossRef][Medline]
21. Pitcher JA, Freedman NJ, Lefkowitz RJ 1998 G protein-coupled receptor kinases. Annu Rev Biochem 67:653–692[CrossRef][Medline]
22. Koenig JA, Edwardson JM 1997 Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci 18:276–287[Medline]
23. Lefkowitz RJ 1998 G protein-coupled receptors: new roles for receptor kinases and ß-arrestins in receptor signalling and desensitization. J Biol Chem 273:18677–18680[Free Full Text]
24. Hunyady L, Catt KJ, Clark AJL, Gáborik Z 2000 Mechanisms and functions of AT₁ angiotensin receptor internalization. Regul Pept 91:29–44[CrossRef][Medline]
25. Walker JKL, Premont RT, Barak LS, Caron MG, Shetzline MA 1999 Properties of secretin receptor internalization differ from those of the ß₂-adrenergic receptor. J Biol Chem 274:31515–31523[Abstract/Free Full Text]
26. Baig AH, Swords FM, Noon L, King PJ, Hunyady L, Clark AJL 2001 Desensitization of the Y1 cell adrenocorticotropin receptor: evidence for a restricted heterologous mechanism implying a role for receptor-effector complexes. J Biol Chem 276:44792–44797[Abstract/Free Full Text]
27. Baig AH, Szaszák M, King PJ, Hunyady L, Clark AJL 2002 Agonist activated adrenocorticotropin receptor internalizes via a clathrin-mediated g protein receptor kinase dependent mechanism. Endocr Res 28:281–289[CrossRef][Medline]
28. Lefkowitz RJ, Cotecchia S, Samama P, Costa T 1993 Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci 14:303–307[CrossRef][Medline]
29. Gether U, Lin S, Kobilka BK 1995 Fluorescent labelling of purified ß₂ adrenergic receptor: evidence for ligand-specific conformational changes. J Biol Chem 270:28268–28275[Abstract/Free Full Text]
30. Lu D, Vage DI, Cone RD 1998 A ligand-mimetic model for constitutive activation of the melanocortin-1 receptor. Mol Endocrinol 12:592–604[Abstract/Free Full Text]
31. Dryja TP 2000 molecular genetics of Oguchi disease, fundus albipunctatus, and other forms of stationary night blindness. Am J Opthalmol 130:547–563
32. Bicknell AB, Lomthaisong K, Woods RW, Hutchinson EG, Bennett HP, Gladwell RT, Lowry PJ 2001 Characterization of a serine protease that cleaves pro--melanotropin at the adrenal to stimulate growth. Cell 105:903–912[CrossRef][Medline]
33. Hornsby PJ 1985 Regulation of adrenocortical cell proliferation in culture. Endocr Res 10:259–281
34. Brown BL, Albano JD, Ekins RP, Sgherzi AM, Tampion W 1971 A simple and sensitive saturation assay method for the measurement of adenosine 3'5'-cyclic monophosphate. Biochem J 121:561–562[Medline]
35. Penhoat A, Jaillard C, Saez JM 1989 Corticotropin positively regulates its own receptors and cAMP response in cultured bovine adrenal cells. Proc Natl Acad Sci USA 86:4978–4981[Abstract/Free Full Text]

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				C Vincent-Dejean, L Cazabat, L Groussin, K Perlemoine, G Fumey, F Tissier, X Bertagna, and J Bertherat Identification of a clinically homogenous subgroup of benign cortisol-secreting adrenocortical tumors characterized by alterations of the protein kinase A (PKA) subunits and high PKA activity. Eur. J. Endocrinol., June 1, 2008; 158(6): 829 - 839. [Abstract] [Full Text] [PDF]

				S. N. Cooray, I. Almiro Do Vale, K.-Y. Leung, T. R. Webb, J. P. Chapple, M. Egertova, M. E. Cheetham, M. R. Elphick, and A. J. L. Clark The Melanocortin 2 Receptor Accessory Protein Exists as a Homodimer and Is Essential for the Function of the Melanocortin 2 Receptor in the Mouse Y1 Cell Line Endocrinology, April 1, 2008; 149(4): 1935 - 1941. [Abstract] [Full Text] [PDF]

				M. G. Teles, S. D.C. Bianco, V. N. Brito, E. B. Trarbach, W. Kuohung, S. Xu, S. B. Seminara, B. B. Mendonca, U. B. Kaiser, and A. C. Latronico A GPR54-Activating Mutation in a Patient with Central Precocious Puberty N. Engl. J. Med., February 14, 2008; 358(7): 709 - 715. [Abstract] [Full Text] [PDF]

				E. Louiset, V. Contesse, L. Groussin, D. Cartier, C. Duparc, G. Barrande, J. Bertherat, H. Vaudry, and H. Lefebvre Expression of Serotonin7 Receptor and Coupling of Ectopic Receptors to Protein Kinase A and Ionic Currents in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia Causing Cushing's Syndrome J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4578 - 4586. [Abstract] [Full Text] [PDF]

				Z. Kilianova, N. Basora, P. Kilian, M. D. Payet, and N. Gallo-Payet Human Melanocortin Receptor 2 Expression and Functionality: Effects of Protein Kinase A and Protein Kinase C on Desensitization and Internalization Endocrinology, May 1, 2006; 147(5): 2325 - 2337. [Abstract] [Full Text] [PDF]

				R. Libe and J. Bertherat Molecular genetics of adrenocortical tumours, from familial to sporadic diseases Eur. J. Endocrinol., October 1, 2005; 153(4): 477 - 487. [Abstract] [Full Text] [PDF]

				F. M. Swords, S. Aylwin, L. Perry, J. Arola, A. B. Grossman, J. P. Monson, and A. J. L. Clark The Aberrant Expression of the Gastric Inhibitory Polypeptide (GIP) Receptor in Adrenal Hyperplasia: Does Chronic Adrenocorticotropin Exposure Stimulate Up-Regulation of GIP Receptors in Cushing's Disease? J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3009 - 3016. [Abstract] [Full Text] [PDF]

				N. Papadopoulou, J. Chen, H. S. Randeva, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Protein Kinase A-Induced Negative Regulation of the Corticotropin-Releasing Hormone R1{alpha} Receptor-Extracellularly Regulated Kinase Signal Transduction Pathway: The Critical Role of Ser301 for Signaling Switch and Selectivity Mol. Endocrinol., March 1, 2004; 18(3): 624 - 639. [Abstract] [Full Text] [PDF]

				Y.-X. Tao and D. L. Segaloff Functional Characterization of Melanocortin-4 Receptor Mutations Associated with Childhood Obesity Endocrinology, October 1, 2003; 144(10): 4544 - 4551. [Abstract] [Full Text] [PDF]

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