Identification of Critical Regions on Phospholipase C-βl Required for Activation by G-proteins

Abstract

In order to determine which portion of phosphoinositide-specific phospholipase C (PLC)-β1 is required for activation by Gαq, a series of specific deletions and truncations of PLC-β1 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 Gαq. The region required for activation was localized to the amino acid sequence corresponding to residues 903-1142 of PLC-β1. 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 Gαq and the other extending from residues Gln-1030 to Leu-1142 that was required for interaction with G α subunits. These results were confirmed by the observation that the C-terminal portion of PLC-β1, when co-expressed with the muscarinic acetylcholine receptor type 1 or the α1C-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-β1 by Gαq. This further localized the sites on PLC-β1 which are involved in interaction with G-protein α subunits.

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