Structure and function of somatostatin receptors in growth hormone control

Transcription

1 S3 Structure and function of somatostatin receptors in growth hormone control I Shimon and S Melmed Department of Medicine, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048, USA (Requests for offprints should be addressed to S Melmed, Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Blvd, B-131, Los Angeles, California 90048, USA) Introduction The two naturally occurring bioactive peptides, somatostatin (SRIF)-14 and SRIF-28 are physiological regulators of pituitary growth hormone (GH), pancreatic endocrine secretions, and gastrointestinal motility and hormone secretion (Brazeau et al. 1972, Mandarino et al. 1981). These biological effects are mediated through specific high-affinity G-protein-coupled receptors containing seven transmembrane domains. Five distinct SRIF receptor (SSTR) subtypes are located on different chromosomes (Bruno et al. 1992, Yasuda et al. 1992, Roher et al. 1993, Xu et al. 1993, Yamada et al. 1992a,b, 1993), and consist of amino acid proteins (39 46 kda) which display 42 60% identity among the different subtypes and 81 97% homology with rodent receptors (Reisine & Bell 1995). The SSTRs interact with different G-proteins (Rens-Domiano et al. 1992, Law et al. 1993) to inhibit adenylate cyclase activity. Receptor subtypes are also associated with other signal-transduction mechanisms, including cationic channel conductance reduction and tyrosine phosphatase activation (Reisine & Bell 1995). The human receptor subtypes 1 4 bind both SRIF-14 and SRIF-28 with high affinity, whereas SSTR5 has preferential affinity for SRIF-28. The clinically available SRIF analogs, octreotide (SMS ) and lanreotide (BIM-23014), bind with high affinity to SSTR2 and have 10-fold less affinity for subtype 5. These analogs effectively suppress GH and thyroid-stimulating hormone (TSH) hypersecretion in patients with GH- and TSH-producing pituitary adenomas (Lamberts et al. 1991). Table 1 SSTR subtype expression in pituitary adenomas. Reproduced from Greenman & Melmed (1996) with the permission of S Karger AG SSTR1 SSTR2 SSRT3 SSTR4 SSTR5 Tumor Non-functioning 1/9 5/9 8/8 0/8 1/8 GH-secreting 3/7 9/10 6/7 0/11 10/11 PRL-secreting 4/5 0/5 3/3 0/3 3/3 ACTH-secreting 1/3 0/3 1/2 0/1 1/1 The normal human pituitary contains SSTR1, 2 and 5, and probably SSTR3 (Miller et al. 1995, Panetta & Patel 1995). All the pituitary hormone-secreting cell types, including those secreting GH, prolactin (PRL), TSH, adrenocorticotropin (ACTH) and gonadotropins express receptors for SRIF (Day et al. 1995, O Carroll et al. 1995). SSTR subtypes in pituitary adenomas Somatostatin-binding sites are present on GH-, TSH- and PRL-secreting and non-functioning adenomas (Reubi & Landolt 1984, 1989, Reubi et al. 1987, Faglia et al. 1991, Bertherat et al. 1992). Octreotide suppression of GH in patients with GH-secreting tumors correlates with tumor SSTR density and with the presence of binding sites demonstrated in vivo by radiolabeled octreotide scan (Reubi & Landolt 1984, 1989). We have demonstrated expression of the different SSTR subtypes in both secreting and non-secreting pituitary adenomas (Greenman & Melmed 1994a,b). Using RNase protection assays and reverse transcriptase (RT)-PCR of RNA samples extracted from pituitary tumor surgical specimens, the expression of four SSTR subtypes was detected in functioning and non-functioning pituitary tumors (Table 1) (Greenman & Melmed 1996). As in the normal human pituitary, SSTR4 was undetectable in all tumors studied. SSTR3 was expressed in the majority of pituitary tumors, regardless of their functional status. SSTR1 expression was observed in less than half of GH-cell adenomas tested (Table 1, Fig. 1). Thus SSTR1, 3 and 4 are probably not involved in hormonal regulation in human pituitary adenoma cells. However, SSTR2, which is predominantly expressed in GH-secreting adenomas (Fig. 1), and SSTR5, which is preferentially detected in secreting adenomas (Fig. 2), might be involved in regulation of secretion of GH and even PRL from adenomatous cells. Recently, others (Miller et al. 1995, Panetta & Patel 1995), using RT-PCR, did not detect SSTR4 in most adenomas, and demonstrated SSTR3 expression in only nine of 47 tumors tested. SSTR2 and 5 were expressed by the majority of the secreting tumors, but they also detected SSTR1 in these secreting tumors (Miller et al. 1995, Panetta & Patel 1995). Journal of Endocrinology (1997) 155, S3 S Journal of Endocrinology Ltd Printed in Great Britain /97/ S3 $08.00/0

2 S4 I SHIMON and S MELMED Pituitary somatostatin receptors SSTR regulation of human fetal pituitary cells Figure 1 SSTR1 and SSTR2 expression in pituitary adenomas. RNA extracted from pituitary tumors was subjected to RNase protection assays using riboprobes for SSTR1 and SSTR2. N, non-functioning adenoma; A, GH-secreting tumor; P, PRL-secreting tumor; C, ACTH-secreting tumor. Adapted from Greenman & Melmed (1994b) The Endocrine Society. Figure 2 SSTR5 expression in pituitary tumors. RNA extracted from pituitary tumors was subjected to RNase protection assay using riboprobe for SSTR5. N, non-functioning adenoma; A, GH-secreting tumor; P, PRL-secreting tumor. Adapted from Greenman & Melmed (1994a) The Endocrine Society. Thus octreotide and lanreotide mediate their hormonal suppression in pituitary adenomatous cells through SSTR2 and possibly SSTR5, which bind these analogs with high affinity (Table 2) and are expressed in the majority of hormone-secreting pituitary adenomas. To elucidate the role of the different SSTR subtypes in regulating pituitary hormone secretion, we recently tested primary human fetal pituitary cells for their responses to SSTR subtype-specific SRIF analogs (Shimon et al. 1997), using cultures derived from week gestation specimens. SSTR2, SSTR5, GH-releasing hormone and insulin-like growth factor-i receptors were shown to be expressed in the fetal specimens. Therefore the normal regulators of pituitary GH secretion are manifest in these cells. The primary pituitary cell culture is a valid in vitro model of functional human pituitary function as, at this gestational age, all hormone-producing cells except for lactotrophs are well differentiated (Asa et al. 1988, 1991). The SRIF analogs depicted in Table 2 may be divided into two groups: those with high affinity for SSTR2 (BIM , , , and also octreotide and lanreotide), and compounds with increased binding affinity for SSTR5 (BIM and ). SRIF regulation of GH and TSH in these fetal cells is mediated by SSTR2 and SSTR5 respectively, as hormonal secretion from fetal pituitary cells was suppressed by analogs with affinity for either SSTR2 or SSTR5 (Fig. 3A). BIM possessed the highest affinity and greatest specificity for human SSTR5 (Coy & Taylor 1996). Interestingly, the structure of BIM differs from that of the other cyclic SSTR analogs in that the disulfide bridge begins and ends at the N- and C-termini of the peptide (positions 1 and 8), rather than positions 2 and 7. Binding affinity for SSTR2 correlated with the analog potency to inhibit GH release (Table 2). Thus SRIF analogs with both comparable and improved efficiency to suppress GH release, including SSTR5-specific analogs, were identified. Interestingly, PRL regulation in the human fetal pituitary is mediated mainly by SSTR2 (Fig. 3A) (Shimon et al. 1997). However, PRL is produced solely by mammosomatotrophs until 24 weeks gestation, and primitive bihormonal stem cells that secrete both PRL and GH (Asa Table 2 Characteristics of SRIF analogs. Adapted from Shimon et al. (1997) by copyright permission of the American Society for Clinical Investigation SSTR binding (IC 50 nm) SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 EC 50 (nm) GH SRIF analog SRIF > > > > > > Octreotide > > ND* Lanreotide > > *ND, not defined. Journal of Endocrinology (1997) 155, S3 S6

3 Pituitary somatostatin receptors I SHIMON and S MELMED S5 Figure 3 Human pituitary hormone suppression (GH, TSH and PRL (A), GH, LH and ACTH (B)) by SRIF analogs exhibiting different SSTR subtype specificity. Human fetal pituitaries were cultured ( cells/well) and 72 h later were treated with different SRIF analogs (10 nm) for 4 h. Each bar represents mean ( S.E.M.) suppression of hormone secretion in four or five wells compared with control wells. L, lanreotide; SRIF, SRIF-14; 23, BIM-23023; 52, BIM-23052; 56, BIM-23056; 68, BIM-23268; 90, BIM-23190; 97, BIM *P<0 05 compared with control (t-test). Adapted from Shimon et al. (1997) by copyright permission of the American Society for Clinical Investigation. et al. 1988) may express different SSTR subtypes from those of mature adult lactotrophs. Several analogs with higher binding affinity for either SSTR2 or SSTR5 also modestly suppressed luteinizing hormone (LH) secretion from human fetal cells (Fig. 3B). In contrast, ACTH secretion was not affected by any SRIF analog tested (Fig. 3B). Journal of Endocrinology (1997) 155, S3 S6

4 S6 I SHIMON and S MELMED Pituitary somatostatin receptors Summary This new generation of SRIF analogs offer exciting opportunities to improve hormone hypersecretion in patients with GH- and TSH-secreting pituitary adenomas and possibly even in patients harboring prolactinomas and non-functioning tumors. Future development of novel analogs with improved affinity for both SSTR2 and SSTR5 may have even greater potency to suppress pituitary hormone hypersecretion and block adenoma growth. Acknowledgements This work was supported by the Doris Factor Molecular Endocrinology Laboratory. References Asa SL, Kovacs K, Horvath E, Losinski NE, Laszlo FA, Domokos I & Halliday WC 1988 Human fetal adenohypophysis: electron microscopic and ultrastructural immunocytochemical analysis. Neuroendocrinology Asa SL, Kovacs K & Singer W 1991 Human fetal adenohypophysis: morphologic and functional analysis in vitro. Neuroendocrinology Bertherat J, Brue T, Enjalbert A, Gunz G, Rasolonjanahary R, Warnet A, Jaquet P & Epelbaum J 1992 Somatostatin receptors on thyrotropin-secreting pituitary adenomas: comparison with the inhibitory effects of octreotide upon in vivo and in vitro hormonal secretions. Journal of Clinical Endocrinology and Metabolism Brazeau P, Vale W, Burgus R, Ling N, Rivier J & Guillemin R 1972 Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science Bruno JF, Xu Y, Song J & Berelowitz M 1992 Molecular cloning and functional expression of a novel brain-specific somatostatin receptor. Proceedings of the National Academy of Sciences of the USA Coy DH & Taylor JE 1996 Receptor-specific somatostatin analogs: correlations with biological activity. Metabolism 45 (Suppl 1) Day R, Dong W, Panetta R, Kraicer J, Greenwood MT & Patel YC 1995 Expression of mrna for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridization analysis. Endocrinology Faglia G, Bazzoni N, Spada A, Arosio M, Ambrosi B, Spinelli F, Sara R, Bonino C & Lunghi F 1991 In vivo detection of somatostatin receptors in patients with functionless pituitary adenomas by means of a radioiodinated analog of somatostatin ([ 123 I]SDZ ). Journal of Clinical Endocrinology and Metabolism Greenman Y & Melmed S 1994a Expression of three somatostatin receptor subtypes in pituitary adenomas. Evidence for preferential SSTR5 expression in the mammosomatotroph lineage. Journal of Clinical Endocrinology and Metabolism Greenman Y & Melmed S 1994b Heterogenous expression of two somatostatin receptor subtypes in pituitary tumors. Journal of Clinical Endocrinology and Metabolism Greenman Y & Melmed S 1996 Somatostatin receptor subtype in pituitary tumors. Frontiers of Hormone Research Lamberts SWJ, Krenning EP & Reubi JC 1991 The role of somatostatin and its analogs in the diagnosis and treatment of tumors. Endocrine Reviews Law S F, Yasuda K, Bell GI&Reisine T 1993 Gia3 and Goa selectively associate with the cloned somatostatin receptor subtype SSTR2. Journal of Biological Chemistry Mandarino L, Stenner D, Blanchard W, Nissen S, Gerish J, Ling N, Brazeau P, Bohlen P, Esch F & Guillemin R 1981 Selective effects of somatostatin-14, -25, and -28 on in vitro insulin and glucagon secretion. Nature Miller GM, Alexander JM, Bikkal HA, Katznelson L, Zervas NT & Klibanski A 1995 Somatostatin receptor subtype gene expression in pituitary adenomas. Journal of Clinical Endocrinology and Metabolism O Carroll A M&Krempels K 1995 Widespread distribution of somatostatin receptor messenger ribonucleic acids in rat pituitary. Endocrinology Panetta R & Patel YC 1995 Expression of mrna for all five human somatostatin receptors (hsstr1 5) in pituitary tumors. Life Sciences Reisine T & Bell GI 1995 Molecular biology of somatostatin receptors. Endocrine Reviews Rens-Domiano S, Law SF, Yamada Y, Seino S, Bell GI & Reisine T 1992 Pharmacological properties of two cloned somatostatin receptors. Molecular Pharmacology Reubi JC & Landolt AM 1984 High density of somatostatin receptors in pituitary tumors from acromegalic patients. Journal of Clinical Endocrinology and Metabolism Reubi J C&Landolt A M 1989 The growth hormone responses to octreotide in acromegaly correlate with adenoma somatostatin receptor status. Journal of Clinical Endocrinology and Metabolism Reubi JC, Heitz PU & Landolt AM 1987 Visualization of somatostatin receptors and correlation with immunoreactive growth hormone and prolactin in human pituitary adenomas: evidence for different tumor subclasses. Journal of Clinical Endocrinology and Metabolism Roher L, Raulf F, Bruns C, Buettner R, Hofstaedter F & Schule R 1993 Cloning and characterization of a fourth human somatostatin receptor. Proceedings of the National Academy of Sciences of the USA Shimon I, Taylor JE, Dong J, Bitonte RA, Kim S, Morgan B, Coy DH, Culler MD & Melmed S 1997 Somatostatin receptor subtype specificity in human fetal pituitary cultures differential role of SSTR2 and SSTR5 for growth hormone, thyroid-stimulating hormone and prolactin regulation. Journal of Clinical Investigation Xu Y, Song H, Bruno JF & Berelowitz M 1993 Molecular cloning and sequencing of a human somatostatin receptor, hsstr4. Biochemical and Biophysical Research Communications Yamada Y, Post SR, Wang K, Medical H, Tager S, Bell GI & Seino S 1992a Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proceedings of the National Academy of Sciences of the USA Yamada Y, Reisine T, Law SF, Ihara Y, Kubota A, Kagimoto S, Seino M, Seino Y, Bell GI & Seino S 1992b Somatostatin receptors, an expanding gene family: cloning and functional characterization of human SSTR3, a protein coupled to adenylyl cyclase. Molecular Endocrinology Yamada Y, Kagimoto S, Kubota A, Yasuda K, Masuda K, Someya Y, Ihara Y, Li Q, Imura H, Seino S & Seino, Y 1993 Cloning, functional expression and pharmacological characterization of a fourth (hsstr4) and fifth (hsstr5) human somatostatin receptor subtype. Biochemical and Biophysical Research Communications Yasuda K, Rens-Domiano S, Breder CD, Law SF, Saper CB, Reisine T & Bell GI 1992 Cloning of a novel somatostatin receptor SSTR3, that is coupled to adenylyl cyclase. Journal of Biological Chemistry Journal of Endocrinology (1997) 155, S3 S6

5 S7 Discussion: Somatostatin receptors Dr P Belchetz (Leeds, UK): I am a little concerned that you are extrapolating from reverse transcriptase polymerase chain reaction (RTPCR) studies and saying that you have expression of the receptor, when there might indeed not be any translation product. Dr S Melmed (Los Angeles, USA): We took great pains to do very careful controls for our RTPCR: all the analogues were tested in an in vitro binding assay, and the analogues were subtype-specific in terms of their in vitro expression; we have expressed all the subtypes in Chinese hamster ovary (CHO) cells and have shown specific binding on CHO cell surfaces, which agree with our pharmacological in vitro data and with the RTPCR. The ultimate answer to your question will come when monoclonal antibodies are available for the specific subtypes, so that we can do double immunostaining on those cells. Until then we are confident that our negative and positive controls and our different in vitro systems are all in correlative agreement. In fact, cell responses to these receptor subtype-selective analogs corroborate the presence of receptor protein. Dr J A H Wass (Oxford, UK): In the very small proportion of patients with acromegaly who are hyperprolactinaemic, octreotide actually suppresses prolactin. Have you any comments on that group? Melmed: We have only two such patients, and it does appear that the acromegalic tissue which also expresses prolactin is more responsive to receptor subtype five-specific analogues than to the native octreotide. That may be clinically very important mixed tumours may respond to a set of analogues different from that to which the pure GH tumours respond. Dr S W J Lamberts (Rotterdam, The Netherlands): So David Coy s 68 analogue might be a promising candidate for long-term suppression of normal GH secretion too. Have you any idea about the pharmacokinetics of this drug? Melmed: All the analogues that I showed you are octapeptides all octreotide analogues. The 68 analogue is structurally somewhat different in that it has two cysteines at Journal of Endocrinology (1997) 155, S7 S Journal of Endocrinology Ltd Printed in Great Britain /97/ S7 $08.00/0

6 S8 Discussion either end it is also linear. We have no idea at all about the in vivo kinetics, but the next few months may tell us. Dr Z Horowitz (Wyoming, USA): Do you see any pattern of receptor subtype expression correlated with the amount of somatostatin that these tumours might be producing? Does that influence the clonal selection of which cells might go on and form the tumour? Melmed: I have no information on that, but it is known that these tumours all make hypothalamic hormones. So that is a natural question to ask: Could the intrapituitary somatostatin ligand be influencing their endogenous receptor? That is a paracrine mechanism that is quite feasible. Journal of Endocrinology (1997) 155, S7 S8