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The human platelet ADP receptor activates Gi2 proteins.

ABSTRACT: We have previously shown that platelet ADP receptors are coupled to G-proteins by measuring the binding of [35S]guanosine-5'-[gamma-thio]triphosphate ([35S]GTP gamma S) to human platelet membranes stimulated with ADP. In order to identify the activated G-proteins, we used an approach which combines photolabelling of receptor-activated G-proteins with 4-azidoanilido-[alpha-32P]GTP and immunoprecipitation of the G-protein alpha-subunits with subtype-specific antibodies. Stimulation of human platelet membranes with ADP resulted in an increase in 4-azidoanilido-[alpha-32P]GTP incorporation into the immunoprecipitates of G alpha i but not of G alpha q proteins, whereas stimulation with the thromboxane analogue U46619 resulted in an increase in 4-azidoanilido-[alpha-32P]GTP incorporation into the immunoprecipitates of G alpha q but not of G alpha i proteins, and thrombin activated both G-proteins. This effect of ADP was concentration dependent and inhibited by the class P2 purinoceptor (P2T) antagonist ATP. Using specific antisera against subtypes of Gi proteins, we found that ADP stimulated labelling of the G alpha 12 immunoprecipitate, but not of the G alpha 13 precipitate. G alpha i1 was not detectable by immunoblotting of platelet membrane proteins. These data suggest that ADP inhibits cAMP formation by activation of G alpha 12 proteins and add evidence in support of the hypothesis that human platelet ADP receptors do not activate PLC through Gq activation.

SUBMITTER: Ohlmann P

PROVIDER: S-EPMC1136181 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Project description:On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.

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The human platelet ADP receptor activates Gi2 proteins.

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