Biomedical subjects

Y C Patel

Publications and source records attributed to Y C Patel.

At least 55 records · Page 3Linked to original sources

PubMed1995

Expression of mRNA for all five human somatostatin receptors (hSSTR1-5) in pituitary tumors.

Expression of mRNA for hSSTR1-5 was determined in secretory (GH, PRL, TSH, ACTH) and nonsecretory pituitary tumors, as well as normal human fetal and adult pituitary by reverse transcriptase (RT) PCR followed by Southern blots. All 5 hSSTR subtype mRNAs were expressed in fetal pituitary, while adult pituitary was positive for 4 subtypes, lacking hSSTR4 mRNA. All 15 tumors analyzed were positive for SSTR mRNA, 14 expressing more than one subtype. SSTR2 mRNA in all tissues was expressed as the 2A variant, there being no detectable transcript for SSTR2B. Amongst the 5 SSTRs, mRNA for SSTR2A was the most frequently expressed (87% of tumors) followed by SSTR1 (73%), SSTR3 (53%), SSTR5 (47%), and SSTR4 (40%). The frequency and pattern of expression of the SSTR mRNAs was virtually identical in the different tumor subclasses and did not correlate with tumor size. Since pituitary tumors are monoclonal in origin, multiple SSTR genes are expressed in individual cells. Most tumors are rich in SSTR1 and SSTR2A mRNA compared to the other subtypes. This implies that SST analogs like SMS 201-995, known to interact with SSTR2A, but not with SSTR1, act on pituitary tumors mainly via the SSTR2 subtype.

Adenoma↗

PubMed1995

The somatostatin receptor family.

The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression, and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR1-4 display weak selectivity for SST-14 binding whereas hSSTR5 is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3, and 5 belong to a similar SSTR subclass. hSSTR1 and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective, and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3-7 which is lined by hydrophobic and charged amino acid residues.

Amino Acid Sequence↗

PubMed1995

Classification and nomenclature of somatostatin receptors.

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR4). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst1, sst2, sst3, sst4, and sst5). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF1) appears to comprise sst2, sst3 and sst5 receptor subtypes. The other class (SRIF2) appears to comprise the other two recombinant receptor subtypes (sst1 and sst4). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

Amino Acid Sequence↗

PubMed1995

Differential stimulation of somatostatin but not neuropeptide Y gene expression by quinolinic acid in cultured cortical neurons.

Somatostatin (SS) and neuropeptide Y (NPY) are coproduced in a subpopulation of neurons that are selectively resistant to NMDA neurotoxicity. We have previously reported that quinolinic acid (QUIN), an NMDA receptor agonist, augments SS mRNA in cultured fetal rat cortical neurons. This study examines coregulation of SS and NPY by QUIN and NMDA in cultured cortical neurons and compares the effects of these agents with those of forskolin and phorbol 12-myristate 13-acetate (PMA), known to activate SS and NPY gene transcription by protein kinase A- and protein kinase C-dependent mechanisms. In addition, transcriptional regulation of the SS gene was investigated by acute transfection of cortical cultures with an SS promoter-chloramphenicol acetyltransferase (CAT) construct. QUIN and NMDA displayed dose-dependent fourfold augmentation of levels of mRNA for SS but not for NPY. In contrast, forskolin and PMA increased both SS and NPY mRNA levels. QUIN- and NMDA-mediated induction of SS mRNA was blocked by the NMDA receptor antagonist (-)-2-amino-5-phosphonovaleric acid and displayed regional brain specificity because it was not observed in fetal hypothalamic cell cultures. In time course studies, the effects of QUIN/NMDA on SS mRNA occurred after a latency of 8 h, indicating a delayed effect. Cortical cells transfected with pSS-750 CAT showed three- to fourfold stimulation of CAT activity with forskolin but not by QUIN or NMDA. These data reveal a dose-dependent, tissue-specific, NMDA receptor-mediated stimulation of SS but not NPY mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Expression of mRNA for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridization analysis.

To characterize cell specific expression of sstr subtypes in the pituitary we have analyzed mRNA for sstr1-5 in rat pituitary somatotrophs by reverse transcriptase polymerase chain reaction and determined the pattern and level of expression of mRNA for sstr subtypes 2 and 5 in individual pituitary cell subpopulations by double label in situ hybridization. Purified somatotrophs expressed mRNA for all 5 sstrs. In situ hybridization analysis revealed sstr5 mRNA in 70% of somatotrophs, 57% of thyrotrophs, 38% of corticotrophs, 33% of lactotrophs, and 21% of gonadotrophs. mRNA for sstr2 occurred in 40% of somatotrophs, 36% of thyrotrophs, 26% of lactotrophs, 3% of corticotrophs, and 8% of gonadotrophs. Not only were more cells positive for sstr5 mRNA but the average number of autoradiographic grains/cell was also higher for sstr5 than sstr2. These results show expression of multiple sstr genes in individual pituitary cells. mRNA for sstr2 and 5 occur in each of the 5 major pituitary cell subsets, sstr5 mRNA being more widely and more abundantly expressed than sstr2.

Animals↗

PubMed1995

Glucocorticoids inhibit somatostatin gene expression through accelerated degradation of somatostatin messenger ribonucleic acid in human thyroid medullary carcinoma (TT) cells.

We previously reported that dexamethasone (DEX) induces dose-dependent biphasic effects on steady state somatostatin (SS) messenger RNA (mRNA) levels in normal rat islet and islet SS-producing tumor cells (1027B2), characterized by stimulation at low doses and marked inhibition at high doses. The stimulatory effect is transcriptionally mediated, whereas the molecular mechanism underlying DEX-induced suppression of SS mRNA levels is unknown. In the present study, we investigated these mechanisms in human thyroid medullary carcinoma (TT) cells, which exhibit only inhibition of SS mRNA with DEX. Cultured TT cells synthesized and secreted large quantities of SS-like immunoreactivity (content, 90 ng/10(6) cells; release, 18 ng/10(6) cells/24h). DEX produced a dose-dependent reduction of both SS-like immunoreactivity secretion and SS mRNA levels, with a maximum inhibition of 60% at 10(-6) M at 48 h. In time-course studies, DEX inhibition of SS function occurred after a lag period of about 12 h, suggesting a posttranscriptional mechanism. To exclude a transcriptional effect of DEX on the SS gene, chloramphenicol acetyltransferase (CAT) activity was determined in TT cells acutely transfected with SS promoter (-750 base pairs) ligated to the receptor CAT gene. No inhibition of CAT activity occurred with DEX (10(-6) M) for 48 h. Furthermore, DEX did not influence the rate of SS gene transcription determined by nuclear run-on assay compared to approximately 2-fold stimulation by cAMP. Actinomycin D (inhibitor of mRNA synthesis) reduced the size of the SS mRNA transcript and rendered it resistant to DEX-induced degradation when coincubated with DEX, but not when it was added after a delay of 12 h, indicating that DEX destabilizes SS mRNA by an active process requiring ongoing gene transcription. Cycloheximide (inhibitor of protein synthesis) reduced SS mRNA levels to the same level as DEX, suggesting that the two agents promote SS mRNA degradation through a common pathway. We conclude that glucocorticoids inhibit steady state SS mRNA levels in TT cells. This effect is not mediated through direct transcriptional inhibition of the SS gene. It requires transcription of another gene(s) whose product(s) accelerates SS mRNA degradation.

Carcinoma, Medullary↗

PubMed1994

Sequence analysis of the 5'-flanking promoter region of the human somatostatin receptor 5.

We have determined the sequence of 2.2 kb of 5' flanking promoter region of the human somatostatin receptor 5 (hsstr5) gene. A number of widely distributed promoter elements were identified including AP1, AP2, AP3, E2A, GCF, and SP1 consensus sequences. hsstr5/CAT gene fusions showed that the 0.9 kb of DNA immediately upstream of the ATG functions as a promoter in rat pituitary GH3 but not in CHO ovary cells. Insertion of this hsstr5 fragment in the anti-sense orientation led to a four fold reduction in CAT activity. Dibutyryl cAMP produced a three fold induction of CAT activity whereas estradiol and retinoic acid had no significant effect. These results indicate that we have identified a DNA fragment at the 5' end of the hsstr5 gene which contains both tissue-specific and regulated elements. The absence of CRE consensus sequence suggests that the cAMP effect is mediated by the multiple AP1 and AP2 sites.

Animals↗

PubMed1994

25-Hydroxycholesterol induces reorganization of lysosomes in normal but not Niemann-Pick disease type C astrocytes.

25-hydroxycholesterol (25-OHC), an oxysterol that potently regulates cellular cholesterol metabolism, induced formation of novel fibrillar structures in normal mouse astrocytes as observed by fluorescence microscopy with the cholesterol probe, filipin. These fibrils were identified as lysosomes by their immunoreactivity for the lysosome associated membrane glycoprotein (LAMP). In contrast, astrocytes derived from the Niemann-Pick disease type C (NPC) mutant mouse were resistant to this oxysterol-induced lysosomal reorganization. NPC astrocytes have abnormal intracellular cholesterol storage as observed by brightly positive filipin staining of their lysosomes. These results show that lysosomal cholesterol storage in NPC astrocytes is associated with a block in oxysterol-mediated fibrillar reorganization of lysosomes.

Animals↗

PubMed1994

Glucocorticoids activate somatostatin gene transcription through co-operative interaction with the cyclic AMP signalling pathway.

The somatostatin (SS) gene is transcriptionally regulated via the cyclic AMP (cAMP) response element (CRE), located in the proximal promoter (-41 to -48 bp). We have previously reported that glucocorticoids induce dose-dependent cell-specific alterations in the steady-state SS mRNA level. Here we have investigated direct transcriptional control of the SS gene by glucocorticoids. We have examined transcriptional interaction between glucocorticoids and the cAMP signalling pathway and mapped the 5' upstream regulatory region of the SS gene involved in glucocorticoid transactivation. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in PC12 rat pheochromocytoma cells and A126-1B2 (protein kinase A-deficient mutant PC12) cells, by acute transfection of 5' flanking SS DNA (- 750, -250 and -71 bp) ligated to the reporter (CAT) gene. Dexamethasone (DEX) induced a dose-dependent 2.2-fold stimulation of SS gene transcription in PC12 cells, but not in A126-1B2 cells. Other steroid and thyroid hormones tested, and retinoic acid, were ineffective, while cAMP and forskolin stimulated gene transcription 4-5-fold in PC12 cells but not in A126-1B2 cells. DEX exerted an additive effect on cAMP-induced gene transcription. Deletion of the promoter from -750 to -71 bp (but not from -750 to -250 bp) abolished all stimulatory effects of DEX without affecting cAMP responsiveness. Mutation of the CRE abrogated both DEX- and cAMP-dependent gene enhancement. Gel electrophoretic mobility shift assays confirmed that the -250 to -71 bp region of the SS promoter (but not the -71 to +55 bp domain) binds specifically to a glucocorticoid response element-sensitive nuclear protein(s) from PC12 cells, suggesting a putative glucocorticoid receptor interaction with SS promoter DNA. We conclude that glucocorticoids regulate SS gene transcription positively. Glucocorticoid-induced transactivation shows dependence on protein kinase. A activity, and may be mediated via protein-protein interaction between the glucocorticoid receptor and the CRE binding protein. DNA sequences upstream from the CRE between -250 and -71 bp in the SS promoter appear to be the target of glucocorticoid action.

Animals↗

PubMed1994

All five cloned human somatostatin receptors (hSSTR1-5) are functionally coupled to adenylyl cyclase.

Recent reports have suggested that only some of the cloned somatostatin receptors (SSTRs) are coupled to adenylyl cyclase. These studies have used both stable and transiently transfected cells or cells lacking appropriate Gi alpha and are controversial. To investigate SSTR signalling mechanisms, we have established stably transfected CHO-K1 cells expressing human genes for SSTR1-5. The effect of 0.1-100 nM SST-14 and SST-28 on forskolin (1 microM) stimulated cAMP accumulation was determined and compared to their receptor binding affinities. The 5 expressed hSSTRs bound SST-14 and SST-28 with high affinity (IC50 1.1-2.1 nM for SST-14; IC50 0.25-5.4 nM for SST-28). hSSTR1-4 bound SST-14 > SST-28 whereas hSSTR5 bound SST-28 > SST-14. Radioligand binding to hSSTR1-5 was significantly inhibited by GTP, GTP gamma S and pertussis toxin. Both SST-14 and SST-28 inhibited forskolin-induced cAMP stimulation with ED50 values which paralleled their binding affinities for the individual hSSTR subtypes. These results demonstrate that all 5 human SSTRs are functionally coupled to inhibition of adenylyl cyclase in CHO-K1 cells via pertussis toxin sensitive G proteins.

Adenylate Cyclase Toxin↗

PubMed1994

Expression of multiple somatostatin receptor genes in AtT-20 cells. Evidence for a novel somatostatin-28 selective receptor subtype.

The pattern of expression of somatostatin receptor (SSTR) genes and gene products in AtT-20 cells was characterized in an attempt to explain the SST-28 binding selectivity that typifies these cells. AtT-20 cells expressed multiple SSTR mRNAs. Paradoxically, this included mRNA for three of the four SST-14 selective receptors: SSTR2 ( +), SSTR1 (+), SSTR4 (+). The SST-28 selective SSTR5 was expressed as a 3.8-kilobase (kb) transcript of relatively low abundance (+) in contrast to normal mouse pituitary which displayed high levels ( ) of a 2.4-kb SSTR5 mRNA. Immunoblot analysis of solubilized membranes with an antipeptide SSTR2 antibody revealed a single SSTR2 protein of 72 +/- 2 kDa. Preincubation of AtT-20 cell membranes with SSTR2 antibody reduced 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding sites by 38%. Residual binding sites exhibited a 4.9-fold increase in affinity for SST-28, a 2.6-fold decrease in affinity for SST-14, and an SST-28:SST-14 potency ratio of 40:1 compared with a potency ratio of 3.5:1 in control membranes. These results demonstrate the expression of four SSTR genes in AtT-20 cells of which SSTR2 predominates. Blockade of SSTR2 with antibody exposes high affinity SST-28 selective sites with comparable binding characteristics to those reported for cloned SSTR5. These SST-28 binding sites may arise from a SSTR5 variant encoded by a high molecular weight 3.8-kb transcript or more likely from another as yet undiscovered member of the SST-28 selective SSTR subfamily.

Amino Acid Sequence↗

PubMed1994

Cysteamine-induced reduction in tissue somatostatin immunoreactivity is associated with alterations in somatostatin mRNA.

The drug cysteamine (CHS) induces a profound loss of somatostatin-14 (SS-14) biological and immunological (SS-14 LI) activity from somatostatin cells in vivo and in vitro. The present study was designed to determine (i) whether CHS induced loss of somatostatin is accompanied by secondary increases in SS-mRNA perhaps through loss of autoinhibition of somatostatin cells; (ii) whether CHS exerts additional direct effects on SS gene regulation. CHS was administered to rats in vivo or applied in vitro to primary cultures of rat islet cells, rat islet somatostatin-producing tumor cells (1027 B2), and endogenous or in vitro synthesized SS-mRNA. In vivo administration of CHS led to 80% reduction in tissue SSLI by 4 h. These changes were accompanied by significant alterations in SS-mRNA that were both tissue-specific and time-dependent. The pattern in brain and intestine was typified by a significant 60% increase in SS-mRNA at 2 h followed by a gradual reduction to approximately 55% of control at 8 h. Stomach showed a significant 95% increase in SS-mRNA at 4 h followed by a 37% decrease by 8 h. Pancreatic SS-mRNA displayed a sustained 25-65% reduction for 8 h. Pretreatment of islet cell cultures with CHS reproduced the in vivo findings with pancreas viz. decreased SSLI (80-90% of control) accompanied by a parallel reduction in SS-mRNA (40-50% of control) sustained from 2-72 h. CHS also induced a reduction in immunoreactive insulin and insulin mRNA in cultured islet cells. As with normal islet cells, CHS treatment of 1027 B2 islet tumor cells led to a profound and sustained decrease in SSLI and SS-mRNA. These changes occurred in the absence of any alteration in intracellular cAMP levels. CHS was without effect when incubated directly with SS-mRNA isolated from 1027 B2 cells or with in vitro synthesized SS-mRNA. We conclude that in addition to its effect on SSLI, CHS also induces time- and tissue-dependent alterations in SS-mRNA. The mechanism of CHS action on SS-mRNA is complex and may involve both an indirect effect secondary to loss of somatostatin autoinhibition (to account for SS-mRNA increases) and/or a direct inhibition of SS gene expression (to explain SS-mRNA reduction). The precise site of direct CHS action on SS gene regulation remains to be defined.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Glucocorticoids regulate somatostatin peptide and steady state messenger ribonucleic acid levels in normal rat tissues and in a somatostatin-producing islet tumor cell line (1027B2).

Although a number of previous studies have suggested that glucocorticoids influence somatostatin (SS) function at the peptide level in some tissues and SS mRNA levels in thyroid tumor cells, there has been no systematic investigation of the effects of glucocorticoids on SS gene expression in normal tissues. In the present study, we have examined the effect of dexamethasone (DEX) on SS secretion and gene expression in rat tissues using as models DEX-treated rats in vivo, primary cultures of rat islet and cerebrocortical cells, and a SS-producing rat islet tumor cell line (1027B2). In vivo DEX administration (0.5 mg/kg) for 3 or 8 days augmented SS-mRNA 2- to 3-fold in peripheral tissues (stomach, pancreas, and jejunum), but reduced it by 50-60% in brain. The DEX effect was time dependent, being more pronounced after 8 days than after 3 days of treatment. In all tissues, SS mRNA levels returned to control values 2 weeks after cessation of DEX. Changes in tissue content of immunoreactive SS paralleled those in SS mRNA. In cultured rat islet cells, 18-h incubation with DEX induced dose-dependent biphasic effects on immunoreactive SS and SS mRNA; low doses (10(-10) M) were stimulatory, and high doses (10(-8)-10(-5) M) were inhibitory. Insulin secretion displayed dose-dependent stimulation by DEX, whereas glucagon release was inhibited. The effect of DEX on SS mRNA levels in primary cultures of brain cells was solely inhibitory. 1027B2 cells responded to DEX with augmented immunoreactive SS secretion and SS mRNA levels at low concentration (10(-10) M), followed by a dose-dependent inhibition of both parameters with increasing DEX concentrations. We conclude that glucocorticoids exert significant effects on SS peptide production and steady state mRNA levels in normal SS-producing tissues in vivo, in vitro, and in cultured 1027B2 cells. The glucocorticoid effect is time and dose dependent, tissue specific, and at least in part due to a direct action of the steroid hormone on SS-producing cells.

Adenoma, Islet Cell↗

PubMed1994

Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hsstr 1-5).

Recent reports (Raynor et al) have claimed the identification of potent somatostatin (SST) agonists exhibiting binding affinities of 1-2 pM and up to 30,000 fold binding selectivity for several of the 5 cloned sstr subtypes. These conclusions, however, are based on binding comparisons of sstr subtypes from different species expressed in different cell lines and studied with different radioligands. To eliminate the effect of species and/or methodological variations, we have investigated agonist selectivity of 32 synthetic SST analogs for all 5 hsstrs stably expressed in CHO-K1 cells under identical binding conditions. We show that hsstr2, 3, 5 react potently with hexapeptide as well as cyclic and linear octapeptide analogs and belong to a similar sstr subclass. hsstr1 and 4 react poorly with these analogs and belong to a separate subclass. The present generation of SST analogs exhibit a modest-50 fold increase in binding potency compared to SST-14 for 2 subtypes (hsstr2, 3), and relative selectivity for only 1 subtype (hsstr2) which is at best only 35 fold. The potency and degree of selectivity of these analogs is several orders of magnitude less than that reported earlier and suggests the need for caution in using these compounds as putative superagonists or subtype selective compounds for any of the individual sstrs.

Binding, Competitive↗

PubMed1994

Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28.

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.

Amino Acid Sequence↗

PubMed1993

A human somatostatin receptor (SSTR3), located on chromosome 22, displays preferential affinity for somatostatin-14 like peptides.

We report here on the cloning of a human intronless gene encoding a member of the G-protein linked somatostatin (SST) receptor subfamily, termed SSTR3. Based on the deduced amino acid sequence, this gene encodes a 418 amino acid protein displaying sequence similarity, particularly within putative transmembrane domains, with the recently cloned human SSTR1 (62%), SSTR2 (64%) and SSTR4 (58%) receptors. Membranes prepared from COS-7 cells transiently expressing the human SSTR3 gene bound [125I]Leu8,D-Trp22,Tyr25 SST-28 in a saturable manner with high affinity (approximately 200 pM) and with rank order of potency (D-Trp8 SST-14 > SST-14 > SMS-201-995 > SST-28) indicative of a somatostatin-14 selective receptor. The pharmacological profile of the expressed human SSTR3 receptor is similar but not identical to that reported for the rat homolog [(1992) J. Biol. Chem. 267, 20422] where the peptide selectivity is SST-28 > or = SST-14 >>> SMS-201-995. Northern blot analysis reveals the presence of an SSTR3 mRNA species of approximately 5 kb in various regions of the monkey brain, including the frontal cortex, cerebellum, medulla, amygdala, with little or no SSTR3 mRNA detectable in brain regions such as the striatum, hippocampus, and olfactory tubercle. The SSTR3 receptor gene maps to human chromosome 22. The existence of at least four distinct human genes encoding somatostatin-14 selective receptors with diverse pharmacological specificities may help to account for some of the multiple biological actions of somatostatin under normal and pathological conditions.

Amino Acid Sequence↗

PubMed1993

Multiple gene transcripts of the somatostatin receptor SSTR2: tissue selective distribution and cAMP regulation.

The rodent SSTR2 mRNA has been reported to be alternatively spliced to generate long (SSTR2A) and short (SSTR2B) receptor isoforms which differ in sequence at their C-terminal regulatory domains. By extending the 3' nucleotide sequence of the human gene (hSSTR2) we show highly conserved intron/exon boundaries suggesting that hSSTR2 is also capable of generating spliced variants. mRNA blots of rat tissues reveal 2 transcripts of 2.8 and 2.3 kb that are differentially expressed in brain regions and multiple peripheral organs. The 2.3 kb mRNA is preferentially expressed in pituitary tumor cells (AtT-20 mouse, GH3 rat, human prolactinoma, human somatotroph adenoma), but not in rat or human insulinoma cells. This transcript shows 4 fold induction by forskolin in AtT-20 cells suggesting cAMP dependent control of SSTR2 gene expression. The abundant expression of SSTR2 gene, the occurrence of 2 isoforms and evidence of extensive regulation at both gene and peptide levels, suggests that SSTR2 is the principal SST-14 selective subtype.

Amino Acid Sequence↗

PubMed1993

Heterologous processing of prosomatostatin in constitutive and regulated secretory pathways. Putative role of the endoproteases furin, PC1, and PC2.

Mammalian prosomatostatin (PSS) is cleaved at a dibasic Arg-Lys site to produce somatostatin-14 (SS-14) and at monobasic Arg and Lys sites to yield SS-28 and PSS(1-10) (antrin), respectively. Furin, PC1, and PC2 are three recently discovered mammalian endoproteases localized either to the constitutive (furin) or regulated (PC1, PC2) secretory pathways. In this study we have compared the heterologous processing of PSS in transiently transfected endocrine (AtT-20 pituitary) and nonendocrine (COS-7 monkey kidney, PC12 pheochromocytoma) tumor cells. We have correlated the efficiency of processing of PSS to SS-14, SS-28, and PSS(1-10) with (i) secretion through the constitutive or regulated pathways; (ii) endogenous expression of mRNA for furin, PC1, and PC2; and (iii) exogenous expression of PC1 and PC2 in cells that do not contain these enzymes in order to delineate the putative role of these enzymes in mediating PSS cleavage at dibasic and monobasic sites and to localize the proteolytic events to specific compartments of the secretory pathways. COS-7 and PC12 cells expressed only furin, secreted constitutively, and processed PSS preferentially at monobasic sites to SS-28 (40-43%) and antrin (27-29%). Processing, however, was inefficient as suggested by large amounts of unprocessed PSS. In contrast, AtT-20 cells showed regulated secretion, expressed all three endoproteases (with high levels of PC1), and processed PSS efficiently to mainly SS-14. PC1, but not PC2, exogenously coexpressed with PSS in COS-7 cells produced significant conversion to SS-14 but not SS-28. This study shows that PSS is capable of monobasic cleavage in the constitutive secretory pathway. Such processing could be mediated by a furin-like enzyme but is relatively inefficient. PC1 can effect dibasic cleavage of PSS whereas PC2 is without influence on PSS processing at least within the constitutive secretory pathway. Although monobasic and dibasic processing of PSS in COS-7 cells correlates with furin-like and PC1 activity, respectively, the relative inefficiency of such processing suggests that compartmentalization of proteolytic events in secretory vesicles or other more specific endoproteases may be required.

Animals↗