seglitide and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

The interaction of the SRIF receptor subtype SSTR2 with pertussis toxin-sensitive G proteins was investigated using an immunoprecipitation approach employing peptide-directed antisera against Gi alpha and G(o) alpha. Antisera directed against either the COOH terminus of Gi alpha or Go alpha uncoupled SSTR2-G protein complexes from CHO cells stably expressing the cloned receptor indicating that both G proteins form complexes with SSTR2. Chinese hamster ovary cells primarily express Gi alpha 3 and G(o) alpha 2 immunoreactivity, with much lower levels of the other pertussis toxin-sensitive G proteins. Antiserum against Gi alpha 3 uncoupled SSTR2/G protein complexes to a similar extent as Gi alpha common antiserum while antisera against Gi alpha 1 and Gi alpha 2 did not. These findings indicate that SSTR2 expressed in Chinese hamster ovary cells predominantly associates with Gi alpha 3 and G(o) alpha 2. In HEK 293 cells which endogenously express low densities of SSTR2 and similar levels of Gi alpha 1 and Gi alpha 3 immunoreactivity but no G(o) alpha, only antiserum directed against Gi alpha 3 immunoprecipitated SSTR2-G protein complexes, indicating that in these cells SSTR2 primarily associates with Gi alpha 3. SRIF can not inhibit forskolin-stimulated cAMP formation in wild-type HEK 293 cells nor in HEK 293 cells transfected with SSTR2. In contrast, SRIF can inhibit cAMP formation in HEK 293 cells expressing the cloned SRIF receptor SSTR3, which requires the presence of Gi alpha 1 to functionally couple to adenylyl cyclase. The lack of efficient association of SSTR2 with Gi alpha 1 may be the cause of its inability to mediate inhibition of cAMP formation. Differences in the G protein-coupling domains of the cloned SRIF receptors may be responsible for their differences in G protein association and ability to effect various signaling pathways.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Blotting, Northern; Blotting, Western; Cell Membrane; CHO Cells; Cholic Acids; Cloning, Molecular; Colforsin; Cricetinae; Cyclic AMP; Detergents; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Kinetics; Mice; Peptides, Cyclic; Pertussis Toxin; Receptors, Somatostatin; Recombinant Proteins; RNA, Messenger; Somatostatin; Transfection; Virulence Factors, Bordetella

Somatostatin (SRIF) induces its biological actions by binding to and stimulating membrane-associated receptors. To investigate the molecular mechanisms by which SRIF induces its biological effects, we have characterized the biochemical properties of SRIF receptors. SRIF receptors can be solubilized in an active form with the detergent CHAPS and can be detected with the high-affinity SRIF analog [125I]MK 678. The pharmacological characteristics of solubilized SRIF receptors from brain are similar to the receptors in membranes, suggesting that the solubilized receptors retain their biological activity. Solubilized SRIF receptors appear to be tightly associated with GTP-binding proteins, since analogs of GTP can greatly reduce agonist labeling of the solubilized SRIF receptor. The solubilized SRIF receptor migrates as a mass of approximately 400 kd and is a glycoprotein since it can specifically interact with lectin columns. The solubilization of the SRIF receptor has allowed for its purification by affinity chromatography. The purified SRIF receptor migrates as a mass of 60 kd in denaturing gels. Using affinity chromatography, the receptor can be purified to near homogeneity. Present studies are directed toward sequencing and cloning cDNA encoding the SRIF receptor in order to further characterize its physical properties and expression.

Topics: Animals; Brain; Cholic Acids; Chromatography, Affinity; Insulin; Membrane Glycoproteins; Peptides, Cyclic; Rats; Receptors, Neurotransmitter; Receptors, Somatostatin; Solubility