G protein G alpha q/11 and G alpha i1,2 are activated by pancreastatin receptors in rat liver: studies with GTP-gamma 35S and azido-GTP-alpha-32P.
In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin-insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using gamma-35S-GTP; specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8-azido-alpha-32P-GTP into liver membranes and into specific immunoprecipitates of different Galpha subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of gamma-35S-GTP in a time- and dose-dependent manner. Activation of the soluble receptors still led to the pancreastatin dose-dependent stimulation of gamma-35S-GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti-Galphaq/11 (85%) and anti-Galphai1,2 (15%) sera, whereas anti-Galphao,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8-azido-alpha-32P-GTP into Galphaq/11 and Galpha, but not into Galphao,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Galphaq/11 and Galphai1,2 proteins. These results suggest that Galphaq/11 and Galphai1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively.
Santos-Alvarez
J
J
Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Hospital Universitario Virgen Macarena, Sevilla, Spain.
Sánchez-Margalet
V
V
D000818:Animals; D001386:Azides / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D010191:Pancreatic Polypeptide / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D011964:Receptors, Gastrointestinal Hormone / Q000502:physiology*
C021961:8-azidoguanosine triphosphate; D001386:Azides; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D011518:Proto-Oncogene Proteins; D011964:Receptors, Gastrointestinal Hormone; C029438:receptors, pancreatic polypeptide-specific; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010191:Pancreatic Polypeptide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C498017:Gnai2 protein, rat; D020962:Heterotrimeric GTP-Binding Proteins