Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.
To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 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 residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.
Kuang
Y
Y
Department of Pharmacology, University of Rochester, NY 14642, USA.
Wu
Y
Y
Smrcka
A
A
Jiang
H
H
Wu
D
D
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007668:Kidney; D046911:Macromolecular Substances; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D015703:Antigens, CD; D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins