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. 1988 Sep 15;254(3):641–647. doi: 10.1042/bj2540641

Molecular characterization of the solubilized receptor of somatostatin from rat pancreatic acinar membranes.

S Knuhtsen 1, J P Esteve 1, B Bernadet 1, N Vaysse 1, C Susini 1
PMCID: PMC1135133  PMID: 2904259

Abstract

The somatostatin receptors on rat pancreatic acinar membranes were demonstrated by use of a radioiodinated (125I-) analogue of somatostatin (SMS 204-090 or [Tyr3]SMS). The tracer was found to bind to the receptor with a Kd of 58 pM. The number of sites detected by this tracer (4.7 pmol/mg of protein) was 5-10 times higher than the number of sites previously found with other tracers. Since the level of non-specific binding was also very low as compared with findings with other tracers, 125I-204-090 might be of interest in future attempts to characterize the somatostatin receptors in the pancreas. The prelabelled membranes were solubilized with 1% CHAPS, and the solubilized complexes were found to adsorb to wheat-germ-agglutinin-coupled agarose, from which they could be eluted with 4 mM-triacetylchitotriose. The complexes within this eluate were shown by gel filtration on Trisacryl GF-2000 to have an Mr of about 400,000. The dissociation of the complexes was augmented both within the membranes as well as in the solubilized state by incubation with the GTP analogue guanosine 5'-[gamma-thio]triphosphate, indicating that the complexes are probably functionally linked to a guanine-nucleotide-binding regulatory protein. After SDS/slab-gel electrophoresis and autoradiography of cross-linked complexes after treatment with the heterobifunctional reagent N-5-azido-2-nitrobenzoyloxysuccinimide, a broad band occurred at approximately Mr 90,000 both in the membranes and in the eluates of complexes after lectin-adsorption chromatography. We conclude that the augmentation of the number of detectable sites for binding of somatostatin, as well as the very low level of non-specific binding obtained by the use of 125I-[Tyr3]SMS as tracer, has made it possible for us to demonstrate the solubilization of the somatostatin receptor in conjunction with its ligand and a GTP-binding regulatory protein, and we have succeeded in cross-linking 125I-[Tyr3]SMS to a binding subunit of Mr 90,000 in the membranes and in demonstrating the presence of the same labelled binding subunit within complexes solubilized and chromatographed on a lectin column before cross-linking.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amsterdam A., Solomon T. E., Jamieson J. D. Sequential dissociation of the exocrine pancreas into lobules, acini, and individual cells. Methods Cell Biol. 1978;20:361–378. doi: 10.1016/s0091-679x(08)62028-2. [DOI] [PubMed] [Google Scholar]
  2. Antoniotti H., Fagot-Revurat P., Esteve J. P., Fourmy D., Pradayrol L., Ribet A. Purification of radioiodinated somatostatin-related peptides by reversed-phase high-performance liquid chromatography. J Chromatogr. 1984 Jul 27;296:181–188. doi: 10.1016/s0021-9673(01)96411-8. [DOI] [PubMed] [Google Scholar]
  3. Brazeau P., Vale W., Burgus R., Ling N., Butcher M., Rivier J., Guillemin R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science. 1973 Jan 5;179(4068):77–79. doi: 10.1126/science.179.4068.77. [DOI] [PubMed] [Google Scholar]
  4. Czernik A. J., Petrack B. Somatostatin receptor binding in rat cerebral cortex. Characterization using a nonreducible somatostatin analog. J Biol Chem. 1983 May 10;258(9):5525–5530. [PubMed] [Google Scholar]
  5. Esteve J. P., Susini C., Vaysse N., Antoniotti H., Wunsch E., Berthon G., Ribet A. Binding of somatostatin to pancreatic acinar cells. Am J Physiol. 1984 Jul;247(1 Pt 1):G62–G69. doi: 10.1152/ajpgi.1984.247.1.G62. [DOI] [PubMed] [Google Scholar]
  6. Koch B. D., Schonbrunn A. The somatostatin receptor is directly coupled to adenylate cyclase in GH4C1 pituitary cell membranes. Endocrinology. 1984 May;114(5):1784–1790. doi: 10.1210/endo-114-5-1784. [DOI] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Lambert M., Svoboda M., Furnelle J., Christophe J. Solubilization from rat pancreatic plasma membranes of a cholecystokinin (CCK) agonist-receptor complex interacting with guanine nucleotide regulatory proteins coexisting in the same macromolecular system. Eur J Biochem. 1985 Mar 15;147(3):611–617. doi: 10.1111/j.0014-2956.1985.00611.x. [DOI] [PubMed] [Google Scholar]
  9. Lewis L. D., Williams J. A. Structural characterization of the somatostatin receptor in rat anterior pituitary membranes. Endocrinology. 1987 Aug;121(2):486–492. doi: 10.1210/endo-121-2-486. [DOI] [PubMed] [Google Scholar]
  10. Liebow C., Hierowski M., duSapin K. Hormonal control of pancreatic cancer growth. Pancreas. 1986;1(1):44–48. doi: 10.1097/00006676-198601000-00009. [DOI] [PubMed] [Google Scholar]
  11. Matozaki T., Sakamoto C., Nagao M., Baba S. Phorbol ester or diacylglycerol modulates somatostatin binding to its receptors on rat pancreatic acinar cell membranes. J Biol Chem. 1986 Jan 25;261(3):1414–1420. [PubMed] [Google Scholar]
  12. Maurer R., Gaehwiler B. H., Buescher H. H., Hill R. C., Roemer D. Opiate antagonistic properties of an octapeptide somatostatin analog. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4815–4817. doi: 10.1073/pnas.79.15.4815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Meldolesi J., Jamieson J. D., Palade G. E. Composition of cellular membranes in the pancreas of the guinea pig. 3. Enzymatic activities. J Cell Biol. 1971 Apr;49(1):150–158. doi: 10.1083/jcb.49.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Redding T. W., Schally A. V. Inhibition of growth of pancreatic carcinomas in animal models by analogs of hypothalamic hormones. Proc Natl Acad Sci U S A. 1984 Jan;81(1):248–252. doi: 10.1073/pnas.81.1.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reubi J. C. New specific radioligand for one subpopulation of brain somatostatin receptors. Life Sci. 1985 May 13;36(19):1829–1836. doi: 10.1016/0024-3205(85)90155-9. [DOI] [PubMed] [Google Scholar]
  16. Reyl-Desmars F., Laboisse C., Lewin M. J. A somatostatin receptor negatively coupled to adenylate cyclase in the human gastric cell line HGT-1. Regul Pept. 1986 Dec 30;16(3-4):207–215. doi: 10.1016/0167-0115(86)90020-0. [DOI] [PubMed] [Google Scholar]
  17. Sakamoto C., Goldfine I. D., Williams J. A. The somatostatin receptor on isolated pancreatic acinar cell plasma membranes. Identification of subunit structure and direct regulation by cholecystokinin. J Biol Chem. 1984 Aug 10;259(15):9623–9627. [PubMed] [Google Scholar]
  18. Sakamoto C., Matozaki T., Nagao M., Baba S. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes. Am J Physiol. 1987 Sep;253(3 Pt 1):G308–G314. doi: 10.1152/ajpgi.1987.253.3.G308. [DOI] [PubMed] [Google Scholar]
  19. Simonds W. F., Koski G., Streaty R. A., Hjelmeland L. M., Klee W. A. Solubilization of active opiate receptors. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4623–4627. doi: 10.1073/pnas.77.8.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Srikant C. B., Patel Y. C. Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes. J Biol Chem. 1986 Jun 15;261(17):7690–7696. [PubMed] [Google Scholar]
  21. Susini C., Bailey A., Szecowka J., Williams J. A. Characterization of covalently cross-linked pancreatic somatostatin receptors. J Biol Chem. 1986 Dec 15;261(35):16738–16743. [PubMed] [Google Scholar]
  22. Toutant M., Aunis D., Bockaert J., Homburger V., Rouot B. Presence of three pertussis toxin substrates and Go alpha immunoreactivity in both plasma and granule membranes of chromaffin cells. FEBS Lett. 1987 May 11;215(2):339–344. doi: 10.1016/0014-5793(87)80174-6. [DOI] [PubMed] [Google Scholar]
  23. Weiland G. A., Molinoff P. B. Quantitative analysis of drug-receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 1981 Jul 27;29(4):313–330. doi: 10.1016/0024-3205(81)90324-6. [DOI] [PubMed] [Google Scholar]

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