Reassembly of phospholipase C-beta2 from separated domains: analysis of basal and G protein-stimulated activities.
Phosphatidylinositol-specific phospholipase C-betas (PLC-betas) are the only PLC isoforms that are regulated by G protein subunits. To further understand the regulation of PLC-beta(2) by G proteins and the functional roles of PLC-beta(2) structural domains, we tested whether the separately expressed amino and carboxyl halves of PLC-beta(2) could associate to form catalytically active enzymes as two polypeptides, and we explored how the complexes thus formed would be regulated by G protein betagamma subunits (Gbetagamma). We expressed cDNA constructs encoding PLC-beta(2) fragments of different lengths in COS-7 cells and demonstrated by coimmunoprecipitation that the coexpressed fragments could assemble and functionally reconstitute an active PLC-beta(2). The pleckstrin homology domain of PLC-beta(2) was required for its targeting to the membrane and for substrate hydrolysis. Reconstituted enzymes that contained the linker region that joins the two catalytic domains were as active or more active than the wild-type PLC-beta(2). When the linker region was removed, basal PLC-beta(2) enzymatic activity was increased further, suggesting that the linker region exerts an inhibitory effect on basal PLC-beta(2) activity. The reconstituted enzymes, like wild-type PLC-beta(2), were activated by Gbetagamma; when the C-terminal region was present in these constructs, they were also activated by Galpha(q). Gbetagamma and Galpha(q) activated these PLC-beta(2) constructs equally in the presence or absence of the linker region. We conclude that the linker region is an inhibitory element in PLC-beta(2) and that Gbetagamma and Galpha(q) do not stimulate PLC-beta(2) through easing the inhibition of enzymatic activity by the linker region.
Zhang
W
W
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. wzhang@calvin.bwh.harvard.edu
Neer
E J
EJ
D020134:Catalytic Domain; D002451:Cell Compartmentation; D015971:Gene Expression Regulation, Enzymologic; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D054799:Phospholipase C beta; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C518036:PLCB2 protein, human; D054799:Phospholipase C beta; D020962:Heterotrimeric GTP-Binding Proteins