Mutations in the carboxyl-terminal domain of phospholipase C-beta 1 delineate the dimer interface and a potential Galphaq interaction site.
The carboxyl-terminal domain of phospholipase C-beta is required for its stimulation by Galpha(q) and for its Galpha(q)-specific GTPase-activating protein (GAP) activity. We subjected this domain to a combination of deletion and alanine/glycine scanning mutagenesis to detect mutations that would inhibit either responsiveness to G(q) or G(q) GAP activity. Most mutations that altered either response or GAP activity diminished both in parallel. Many of these mutations map at the interface at which the carboxyl-terminal domain was recently shown to form a dimer (Singer, A. U., et al. (2001) Nat. Struct. Biol., 9, 32-36). Most others clustered in an area that is a plausible Galpha(q) binding site. In addition, one mutation that differentially inhibited GAP activity relative to responsiveness to Galpha(q) mapped in this region at a location modeled to be in close contact with the switch II region of Galpha(q). This is the site at which RGS proteins are thought to exert their GAP activity. Last, a deletion mutation differentially inhibited the response of phospholipase C-beta1 to Galpha(q) without blocking GAP activity. Its location in the molecule suggests that moving the attachment point of the catalytic domain can disrupt its ability to be activated by Galpha(q).
Ilkaeva
Olga
O
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041.
Kinch
Lisa N
LN
Paulssen
Ruth H
RH
Ross
Elliott M
EM
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D054799:Phospholipase C beta; D011487:Protein Conformation; D017384:Sequence Deletion; D018411:Spodoptera; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins