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.
Wu
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Division of Biology, California Institute of Technology, Pasadena 91125.
Jiang
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Katz
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Simon
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D000595:Amino Acid Sequence; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009638:Norepinephrine / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
D007295:Inositol Phosphates; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D002217:Carbachol; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D009638:Norepinephrine