Phospholipase C-β3 and -β1 Form Homodimers, but Not Heterodimers, through Catalytic and Carboxyl-Terminal Domains

Abstract

Phospholipase C-␤ (PLC-␤) isoenzymes are key effectors in G protein-coupled signaling pathways. Prior research suggests that some isoforms of PLC-␤ may exist and function as dimers. Using coimmunoprecipitation assays of differentially tagged PLC-␤ constructs and size-exclusion chromatography of native PLC-␤, we observed homodimerization of PLC-␤3 and PLC-␤1 isoenzymes but failed to detect heterodimerization of these isoenzymes. Size-exclusion chromatography data suggest that PLC-␤3 and PLC-␤1 form higher affinity homodimers than PLC-␤2. Evidence supportive of limited PLC-␤ monomerhomodimer equilibrium appears at Յ100 nM. Further assessment of homodimerization status by coimmunoprecipitation assays with differentially tagged PLC-␤3 fragments demonstrated that at least two subdomains of PLC-␤3 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-␤ homodimers in a whole-cell context, using fluorescent protein-tagged constructs and microscopic fluorescence resonance energy transfer assays.Phospholipase C (PLC) is a key effector enzyme in multiple cellular signaling pathways, hydrolyzing phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate and diacylglycerol, second messengers that subsequently regulate Ca 2ϩ release from intracellular stores and protein kinase C activation. PLC isozymes are grouped into five families based on structure and regulatory mechanism: PLC-␤, PLC-␥, PLC-␦, PLC-, and PLC-. The PLC-␤ family exists as four known homologs in mammals, ␤1-␤4, all of which are involved in G protein-coupled receptor-mediated signaling cascades (Rhee, 2001;Saunders et al., 2002). Of the four isoenzymes, PLC-␤1 and PLC-␤3 are widely distributed and are often found coexpressed in a variety of tissues, including brain, liver, uterus, parotid gland, lung, platelets, and heart (Hansen et al., 1995;Fukami, 2002).PLC-␤ isoenzymes contain sequences with homology to several characterized protein structural motifs commonly found in membrane associated, Ca 2ϩ and lipid binding proteins. These structural domains include pleckstrin homology (PH), EF hand, and C2 homology regions, as well as a PDZbinding domain at the C terminus. Highly conserved among PLC family members are two domains required for catalytic activity, designated X and Y, that structurally form a triose phosphate isomerase barrel (TIM) to create the catalytic pocket in the crystal form of PLC-␦ (Rhee, 2001).Unique among PLC family members, PLC-␤ isoenzymes contain a carboxyl-terminal (C-tail) domain of approximately 400 amino acids that is involved in membrane association and regulation by G␣ q subunits. Crystallization of the isolated C-tail domain of a turkey PLC-␤ revealed a structure of three ␣-helices forming a coiled coil that associates as an antiparallel homodimer along its long axis (Singer et al., 2002). Size-exclusion chromatography data suggested that purified, concentrated, f...

Structural Insights into Phospholipase C-β Function

“…Mutation of conserved hydrophobic residues within the analogous dimer interface of PLCb1 were shown to impair activation by Ga q (Ilkaeva et al, 2002), and size exclusion analysis of both purified PLCb proteins and isolated distal CTDs, as well as cell-based studies, suggested the existence of dimers (Singer et al, 2002;Zhang et al, 2006). Conversely, studies of fulllength human PLCb3 found no evidence of oligomerization as assessed by size exclusion chromatography, multiangle light scattering, cryo-electron microscopy, or X-ray crystallography (Lyon et al, 2013).…”

“…The "core" of the domain, which contains some of the most highly conserved residues, is found where the Da2 helix crosses one face of the helical bundle. The entire distal CTD is stabilized primarily through coiled-coil interactions, which may have relatively low stringency for amino acid side chains, and thus could account for the low sequence conservation (Singer et al, 2002;Zhang et al, 2006;Lyon et al, 2013). The tertiary Fig.…”

PI(3,4,5)P3potentiates phospholipase C-β activity

“…1321N1, H9c2 and HEK 293 cells were cultured in 90% DMEM, 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C under 5% CO 2 in humidified air [6]. One day prior to transfection, HEK 293 cells were plated at a density of 5×10 5 cells/10 cm culture dish.…”

“…The central XY domain is highly conserved among PLC isozymes and structurally forms a catalytic TIM ( T riosephosphate i so m erase) barrel to create the catalytic pocket for PI(4,5)P 2 binding [4]. PLC-β isozymes are distinguished by a structurally unique C-tail domain (approximately 400 amino acids) that is involved in regulation by Gα q subunits [1, 5] and dimerization [6-8]. …”

Regulating G protein activity by lipase-independent functions of phospholipase C

“…Nonetheless, homodimerization of the avian full length coiled-coil domain, the deletion avian coiled-coil domain, fulllength avian PLC-β and PLC-β 1 was corroborated by size exclusion chromatography. A second study reported that full length PLC-β 1 and PLC-β 3 homodimerized, dependent on the coiled-coil domain [114]. Dimerization was validated by size exclusion chromatography and coimmunoprecipitation.…”