Molecular characterization of the solubilized receptor of somatostatin from rat pancreatic acinar membranes.
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.
Knuhtsen
S
S
Groupe de Recherche de Biologie et Pathologie Digestive, I.N.S.E.R.M. U 151, CHU Rangueil, Toulouse, France.
Esteve
J P
JP
Bernadet
B
B
Vaysse
N
N
Susini
C
C
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D015282:Octreotide / Q000031:analogs & derivatives / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D013873:Thionucleotides; C045210:3-Tyr-octreotide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; D006160:Guanosine Triphosphate; D015282:Octreotide