Fish somatostatin sst3 receptor: comparison of radioligand and GTPγS binding, adenylate cyclase and phospholipase C activities reveals different agonist‐dependent pharmacological signatures
Authors: Siehler S. , Nunn C. , Zupanc G. K. H. , Hoyer D. 
Published: January 2005

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non‐mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPγS binding, inhibition of forskolin‐stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5).

2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G‐protein‐coupled fsst3 receptors. By contrast, [125I]LTT‐SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non‐coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT‐SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg−1) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg−1).

3 SRIF14 (10 μm)‐stimulated specific [35S]GTPγS binding by three‐fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60–80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin‐stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five‐ to six‐fold, as determined by measuring total [3H] IPx accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml−1, 21% inhibition), which suggests the Gq‐family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20–70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14‐induced total [3H]‐IPx production (pKB = 6.83), but failed to block competitively agonist‐stimulated [35S]GTPγS binding or agonist‐induced inhibition of FSAC activity.

4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPγS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410–0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPγS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT‐SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75–0.83; 0.68–0.89). By contrast, the PLC stimulation and radioligand‐binding profiles did not correlate.

5 Comparison of the functional data (GTPγS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17).

6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp‐sensitivities determined with the four radioligands (agonists) are assumed to results from ligand‐specific states of the fsst3‐ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist‐induced receptor trafficking.