IDENTIFICATION OF A PHOSPHOLIPASE-C BETA(2) REGION THAT INTERACTS WITH G-BETA-GAMMA

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta(2) sequences i nvolved in interactions with the beta gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta(2) segments that span the region from the beginning of the X box to the end of the Y box, We found the sequence extending from r esidue Glu-435 to residue Val-641 inhibited G beta gamma-mediated acti vation of PLC beta(2) in transfected COS-7 cells, This PLC beta(2) seq uence also inhibited ligand-induced activation of PLC in COS-7 cells c otransfected with cDNAs encoding the complement component C5a receptor and PLC beta(2) but not in cells transfected with the alpha(1)B-adren ergic receptor, suggesting that the PLC beta(2) residues (Glu-435 to V al-641) inhibit the G beta gamma-mediated but not the G alpha-mediated effect. The inhibitory effect on G beta gamma-mediated activation of PLC beta(2) may be the result of the interaction between G beta gamma and the PLC beta(2) fragment. This idea was confirmed by the observati on that a fusion protein comprising these residues (Glu-435 to Val-641 ) of PLC beta(2) and glutathione S-transferase (GST) bound to G beta g amma in an rn vitro binding assay, The G beta gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta(2) sequences a nd GST in the in vitro binding assay.