Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins.

In order to determine which portion of phosphoinositide-specific phospholipase C (PLC)-beta 1 is required for activation by G alpha q, a series of specific deletions and truncations of PLC-beta 1 cDNA were prepared. After transfection of COS-7 cells with these cDNA clones, the activity and localization of the expressed proteins were determined. Specific deletions in the C-terminal end of the protein did not lead to loss of intrinsic enzymatic activity but did result in loss of the ability to be activated by G alpha q. The region required for activation was localized to the amino acid sequence corresponding to residues 903-1142 of PLC-beta 1. This region was further subdivided into two sequences; one extending from residues Thr-903 to Gln-1030 that was required for particulate fraction association as well as for activation by G alpha q and the other extending from residues Gln-1030 to Leu-1142 that was required for interaction with G alpha subunits. These results were confirmed by the observation that the C-terminal portion of PLC-beta 1, when co-expressed with the muscarinic acetylcholine receptor type 1 or the alpha 1C-adrenergic receptor in COS-7 cells, markedly inhibited ligand-induced release of inositol phosphates. In an in vitro system, two peptides derived from the G-protein interaction region at the C terminus were found to inhibit the guanosine 5'-3-O-(thio)triphosphate-dependent activation of PLC-beta 1 by G alpha q. This further localized the sites on PLC-beta 1 which are involved in interaction with G-protein alpha subunits.