title: Characterisation of human recombinant somatostatin receptors. 2. Modulation of GTPgammaS binding.
abstract: G protein activation by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and analogues of these neuropeptides was investigated at human somatostatin receptor subtypes 1-5 (sst1-5) stably expressed in CCL39 Chinese hamster lung fibroblast cells by measuring agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. [35S]GTPgammaS binding was assessed in the presence of 100 mM NaCl and 1 microM GDP, although higher Emax and/or pEC50 values may have been obtained under other conditions, but at the expense of lower absolute stimulation or signal/noise ratio. SRIF14 stimulated [35S]GTPgammaS binding to 162, 220, 148 and 266% of control levels via sst2, sst3, sst4 and sst5 receptors, respectively. At sst1 receptors, SRIF14 produced only a limited stimulation (Emax 115%). Hence sst1 receptors were not subjected to further [35S]GTPgammaS binding experiments. [35S]GTPgammaS binding assays were then performed with sst2-5 receptors. Most of the peptide analogues stimulated [35S]GTPgammaS binding in sst2-5 receptor-expressing cells. BIM 23056 behaved as an antagonist on SRIF14-induced [35S]GTPgammaS binding with an apparent pKBs of 6.33 and 5.84 at hsst3 and hsst5 receptors respectively, whereas neither agonism nor antagonism could be shown (at 1 microM) at sst2 or sst4 receptors. The effect at sst5 receptors was not surmountable and needs further investigations. The so-called "antagonist" SA, was devoid of antagonist activity at sst2 or sst3 receptors, whereas it was almost a full agonist at sst4 and sst5 receptor-mediated [35S]GTPgammaS binding. The [35S]GTPgammaS-binding profiles of hsst2-5 receptors were compared with their respective radioligand binding profiles. For sst4 and sst5 receptors, the rank order of affinity of all tested radioligands correlated highly significantly with [35S]GTPgammaS binding (r = 0.814-0.897). At sst3 receptors, [35S]GTPgammaS binding correlated somewhat less with binding profiles obtained with [125I][Tyr10]CST14 and [125I]CGP 23996 than with [125I]LTT-SRIF28 (r = 0.743, 0.757 and 0.882, respectively). At sst2 receptors, [35S]GTPgammaS binding correlated with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I][Tyr3]octreotide binding profiles (r = 0.596-0.699), but not with [125I][Tyr10]CST14 binding. The present [35S]GTPgammaS binding data combined to previous radioligand binding results obtained in cells expressing human SRIF receptors, suggest that at any given receptor, agonists' rank orders of potency (not to mention absolute affinity values which vary profoundly) are not as strictly ordered as may be anticipated. We are investigating these aspects further by analysing additional signalling pathways.
authors.affiliation: Nervous System Research, Novartis Pharma AG, Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Siehler
authors.forename: D
authors.initials: D
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000166:cytology; D011869:Radioligand Assay; D017481:Receptors, Somatostatin / Q000819:agonists / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism; D014162:Transfection
chemical_list: D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin