Bombesin activation of phospholipase C β 1 in rat acinar pancreatic cells involves the pertussis toxin-sensitive G αi3 protein

Abstract

Bombesin stimulation of inositol 1,4,5-trisphosphate (Ins P 3) formation in rat sonicated pancreatic acinar cells was inhibited by an antibody directed against the pertussis toxin (PTX)-sensitive GTP-binding G αi3 protein but not by an anti-G αq-11 antibody. After solubilization and gel filtration, [ 125I-Tyr 4]bombesin binding sites were recovered in a peak of protein of 67 ∼ 90 kDa with a maximal enrichment corresponding to a molecular mass of 83-kDa. Results obtained from the non-hydrolysable GTP analog guanosine-5′-[γ-thio]triphosphate (GTPγS) binding, PTX-stimulated ADP-ribosylation and immunoblotting showed that the 83-kDa fraction contained the G αi3 protein but not the G αq-11 protein. Furthermore, GTPγS increased the bombesin binding dissociation constant ( K D ) from 0.32 to 0.60 nM, while the anti-G αi3 antibody decreased the maximal binding capacity ( B max ) from 50 to 25 fmol/mg protein without affecting the K D . Mixing solubilized bombesin binding sites with a phospholipase C (PLC) preparation from rat pancreas reconstituted a bombesin-stimulated PLC activity which was markedly inhibited by the anti-G αi3 antibody but unaffected by the anti-G αq-11 antibody. In addition, this stimulation was inhibited by an anti-PLC β 1 antibody. This result supports the involvement of the PLC β 1 isoform in bombesin receptor activation.