Abstract
Phospholipase C-β (PLC-β) isoenzymes are key effectors in G protein-coupled signaling pathways. Prior research suggests that some isoforms of PLC-β may exist and function as dimers. Using coimmunoprecipitation assays of differentially tagged PLC-β constructs and size-exclusion chromatography of native PLC-β, we observed homodimerization of PLC-β3 and PLC-β1 isoenzymes but failed to detect heterodimerization of these isoenzymes. Size-exclusion chromatography data suggest that PLC-β3 and PLC-β1 form higher affinity homodimers than PLC-β2. Evidence supportive of limited PLC-β monomer-homodimer equilibrium appears at ≤100 nM. Further assessment of homodimerization status by coimmunoprecipitation assays with differentially tagged PLC-β3 fragments demonstrated that at least two subdomains of PLC-β3 are involved in dimer formation, one in the catalytic X and Y domains and the other in the G protein-regulated carboxyl-terminal domain. In addition, we provide evidence consistent with the existence of PLC-β homodimers in a whole-cell context, using fluorescent protein-tagged constructs and microscopic fluorescence resonance energy transfer assays.
Footnotes
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This work was supported by the Pharmaceutical Research and Manufacturer's Association Foundation (to T.M.F.), the American Foundation for Pharmaceutical Education (J.S.M.), an Oregon Sports Lottery Scholarship (to Y.Z.), and the National Institutes of Health, National Institute of General Medical Sciences grant R01-GM61244 (to T.M.F.). This publication was made possible, in part, by the Confocal Microscopy Facility of the Environmental Health Sciences Center at Oregon State University, with funding from the National Institute of Environmental Health Sciences, National Institutes of Health grants P30-ES00210 and 1S10-RR107903-01.
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ABBREVIATIONS: PLC, phospholipase C; PH, pleckstrin homology; SEC, size-exclusion chromatography; FRET, fluorescence resonance energy transfer; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; CFP, cyan fluorescent protein; PCR, polymerase chain reaction; YFP, yellow fluorescent protein; aa, amino acid(s); HEK, human embryonic kidney; His6, hexahistidine; TCA, trichloroacetic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloroacetic acid; PAGE, polyacrylamide gel electrophoresis; TCEP, tris(2-carboxyethyl) phosphine hydrochloride.
- Received December 28, 2005.
- Accepted June 8, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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