Characterisation of human recombinant somatostatin receptors. 2. Modulation of GTPγS 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 (sst_1–5) stably expressed in CCL39 Chinese hamster lung fibroblast cells by measuring agonist-stimulated [³⁵S]guanosine 5'-O-(3-thiotriphosphate) ([³⁵S]GTPγS) binding. [³⁵S]GTPγS binding was assessed in the presence of 100 mM NaCl and 1 µM GDP, although higher E _max 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]GTPγS 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 (E _max 115%). Hence sst₁ receptors were not subjected to further [³⁵S]GTPγS binding experiments. [³⁵S]GTPγS binding assays were then performed with sst_2–5 receptors. Most of the peptide analogues stimulated [³⁵S]GTPγS binding in sst_2–5 receptor-expressing cells. BIM 23056 behaved as an antagonist on SRIF₁₄-induced [³⁵S]GTPγS binding with an apparent pK _Bs of 6.33 and 5.84 at hsst₃ and hsst₅ receptors respectively, whereas neither agonism nor antagonism could be shown (at 1 µM) 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]GTPγS binding. The [³⁵S]GTPγS-binding profiles of hsst_2–5 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]GTPγS binding (r=0.814–0.897). At sst₃ receptors, [³⁵S]GTPγS 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]GTPγS 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]GTPγS 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.