Characterization of covalently cross‐linked somatostatin receptors in hamster beta cell insulinoma

Abstract

The selective binding of somatostatin-28 (SS-28) to beta cells of hamster insulinoma was characterized using HPLC-purified '251-[Le~8,~-Trp22,Tyr25]SS-28 or lZ5I-SS-28. A single class of high-affinity sites (Kd = 53 f 5 pM) was observed with a binding capacity of 2.85 pmol/mg membrane protein. A large number of relatively low-affinity sites was found also. The order of potency of different peptides to inhibit '251-SS-28 binding is SS-28 > SS-14 > SMS-201-995 and the respective half-maximal inhibitory doses are 0.16 nM, 10 nM and 1000 nM. CCK8 and other active pancreatic peptides (glucagon, insulin, gastric inhibitory peptide, vasoactive intestinal peptide, oxyntomodulin) do not inhibit the SS-28 receptor binding. '251-SS-28-labeled beta membranes were successfully cross-linked using either the cleavable cross-linker dithiobis(succinimidy1propionate) (1 mM) alone or with a heterobifunctional agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB). In both cases five molecular components were revealed, after polyacrylamide gel electrophoresis of the membrane proteins and autoradiography, with the following molecular mass: 196-kDa, 132 kDa, 69 kDa, 45 kDa and 28 kDa. The labeling of 196-kDa, 132-kDa and 45-kDa species was specific in that they could be inhibited by unlabeled SS-28. The major labeled species corresponds to the 132-kDa band and no change in the mobility of this HSAB covalently bound SS-28 receptor was found after addition of dithiothreitol, suggesting that this specific receptor does not contain interchain disulphide bonds. The molecular mass of SS-28 receptors differs markedly from that of guineapig pancreatic acinar membranes, where a single 93-kDa protein is identified as a '251-SS-28 receptor site in comparative experiments. Both the binding kinetics and structural differences sustain the selective action of SS-28 in the endocrine pancreas.Somatostatin (SS), or somatotropic release inhibiting factor, was initially isolated from the ovine hypothalamus [l]. is mostly abundant in gut epithelium whereas somatostatin-14 [3] is mainly present in D cells of islets of Langerhans [3] and appears early in the developmental mammals [4]. Both inhibit, at different concentrations, the release of insulin and glucagon in vitro and in vivo [5, 61. The mechanism by which the somatostatin initiates the cellular response is the binding of somatostatin to specific receptors as shown in different tissues (reviewed in [7]). The somatostatin receptors have also been found in isolated rat islets of Langerhans [XI and in membrane of hamster insulinoma [9]. This insulinoma represents an invaluable model for somatostatin receptor studies, since it is composed of mainly beta cells [lo] and an array of other regulatory receptors (glucagon, oxyntomodulin, gastric inhibitory peptide) related to the beta cell function [ll -131 (reviewed in [14]). Furthermore, in this tumor the glucose-induced insulin release is inhibited by suggesting the presence of functional somatostatin receptors in these beta cells. Whereas the understandi...

Solubilization of somatostatin receptors in hamster pancreatic beta cells

“…In our previous report [12], specific somatostatin-28-receptor complexes of 196 kDa, 132 kDa and 45 kDa were idcntified by direct cross-linking of 1251-somatostatin-28 bound to mem brane-embedded receptors. We now report that solubilization of the p cell mcmbranes with the nondenaturing detergent Triton X-100 results in one major somatostatin-28 receptor species of 128 kDa and two minor species of 228 kDa and 45 kDa after cross-linking and SDS/ PAGE analysis.…”

“…Indeed, differences in ligand specificity and in functional properties of thc somatostatin receptors in these tissues have been noted. In the hamster insulinoma, the somatostatin receptors have a higher affinity for somatostatin-28 than somatostatin-14 [12,191. However somatostatin-14 is able to inhibit insulin release [19] although less effectivcly than somatostatin-28.…”

“…The cross-linking of '251-somatostatin-28 labelled membranes with dithiobis(succinimidy1 propionate) was performed as described previously [12]. Briefly, '251-somatostatin-28-bound mcmbranes (50 pg protein) were resuspended in 1 ml 60 mM Hepes pH 7.5 and the cross-linking reaction was performed with 1 mM DSP at 4'C.…”

“…Cyclic AMP has been assigned a regulatory role in pancreatic beta cells [lo] and experimentally induced type I diabetes has been shown to be associated with the loss of the inhibitory activity of G, on adenylate cyclase [ll]. In order to elucidate the critical involvement of the somatostatin receptor in the membranesignalling process, knowledge of the structural composition of the somatostatin receptors and an understanding of the interaction between the somatostatin receptor and GTP-binding proteins are necessary.Recently, we have identified, by direct cross-linking of '25J-somatostatin-28 to hamster pancreatic beta cells, three specific somatostatin-28 receptor complexes of 196 kDa, 132 kDa and 45 kDa which are different from that of pancreatic acinar cell [12]. The presence of interchain disulphide bonds was not observed with the major 132-kDa species.…”

“…Recently, we have identified, by direct cross-linking of '25J-somatostatin-28 to hamster pancreatic beta cells, three specific somatostatin-28 receptor complexes of 196 kDa, 132 kDa and 45 kDa which are different from that of pancreatic acinar cell [12]. The presence of interchain disulphide bonds was not observed with the major 132-kDa species.…”