Pharmacological characterisation of the goldfish somatostatin sst 5 receptor

Abstract

Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst 1–5). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst 5 receptors (gfsst 5) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [ 125I]-LTT-SRIF-28 ([Leu 8, dTrp 22, 125I-Tyr 25]SRIF-28) and [ 125I][Tyr 10]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (p K d: 9.99–9.71; B max: 300–350 fmol mg −1). 5′-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5–57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [ 125I]LTT-SRIF-28 and [ 125I][Tyr 10]cortistatin-14 correlated significantly ( r 2=0.97, n=20). Pharmacological profiles of human and mouse sst 5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst 5 profile ( r 2=0.52–0.78, n≥16). Functional expression of the gfsst 5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [ 35S]GTPγS ([ 35S]guanosine 5′- O-(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies ( r 2=0.81–0.93, n=20), although relative potency (pEC 50) was reduced compared to p K d values. Relative efficacy profiles of luciferase expression and [ 35S]GTPγS binding, were rather divergent ( r 2=0.48, n=20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 ( d-Phe-Phe-Tyr- d-Trp-Lys-Val-Phe- d-Nal-NH 2) acted as an antagonist on the effects of SRIF-14 (p K B=6.74±0.23) on stimulation of [ 35S]GTPγS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [ 35S]GTPγS binding suggesting coupling of the receptor to G i/G o proteins. In summary, the present studies demonstrate that the gfsst 5 receptor has a similar pharmacological profile and transductional properties to mammalian sst 5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.