Regulatory Interactions between the Amino Terminus of G-protein βγ Subunits and the Catalytic Domain of Phospholipase Cβ2

Abstract

Activation of G-protein-coupled receptors releases the G␤␥ subunit from G␣-GTP, leaving G␤␥ free to interact with effector molecules such as enzymes or ion channels (1-3). A particular group of enzymes regulated by G␤␥ belong to the phosphoinositide-specific phospholipase C␤ (PLC␤) 1 class. Once activated, PLC␤ cleaves phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into the two second-messengers, diacylglycerol and inositol 1,4,5-trisphosphate. All PLC␤ isoforms have an N-terminal pleckstrin homology (PH) domain, four sets of EF-hand domains, X and Y domains comprising the catalytic center, and a C2 domain followed by a C-terminal extension (4). There are four isoforms of PLC␤, ␤1-4; however, only PLC␤2 and PLC␤3 are regulated by ␤␥ subunits (5).G-protein ␤ subunits have two distinct domains, an N-terminal ␣ helix, which forms a coiled-coil interaction with the ␥ subunit, and a seven-blade ␤-propeller structure composed of seven WD repeating motifs (6, 7). Amino acids within blades one, two, and three make extensive contacts with ␣-GDP (7, 8). Since ␣ subunits block G␤␥-dependent regulation of all effectors, effector-binding sites on G␤␥ were predicted to overlap with the ␣ subunit binding site. Indeed, mutagenesis of particular amino acids on G␤␥ important for ␣ subunit binding blocked activation of PLC␤2 (9, 10). Furthermore, mutations to the outer strands of blades 2, 6, and 7, which do not make direct contact with G␣, rendered G␤␥ unable to effectively activate PLC␤2 (11).The mechanism for regulation of PLC␤2 by G␤␥ subunits is not fully understood. Approaches for elucidating the activation mechanism have been to characterize the effects of a variety of mutants, peptides, and fusion proteins on regulation of PLC␤2 by G␤␥. Two major regions on PLC␤2 have been implicated as G␤␥-binding sites, the N-terminal PH domain and a portion of the catalytic Y domain. A large body of evidence supports a role for interactions between the catalytic domain of PLC␤2 and G␤␥ subunits. Overexpression of the Y domain in COS-7 cells blocked receptor-mediated G␤␥-dependent, but not G␣ qdependent activation of PLC. A purified glutathione S-transferase fusion protein comprising amino acids 526 -641 of the Y-domain bound directly to purified G␤␥ subunits (12). Overlapping peptides representing a portion of the Y-domain directly cross-linked to both G␤ and G␥ subunits with the heterobifunctional cross-linker SMCC, and this was blocked by purified PLC␤2 but not ␣ i . The peptides also inhibited G␤␥ regulation of PLC␤2 with an EC 50 of 30 -50 M (13). The crystal structure of PLC␦ reveals that the region implicated by the peptide studies corresponds to the ␣ 5 helix on the surface of the catalytic domain and therefore is accessible to interact with G␤␥ subunits (14). We hypothesized that direct binding of this helix within the catalytic domain to G␤␥ is involved in G␤␥-dependent PLC␤2 activation.To define the site cross-linking of one of the catalytic domain peptides, N20K (amino acids 565-574 in PLC␤2), to the G␤ subunit and identify a...

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…Additional regions found to be important for the Gβγ -activation of PLC-β2 (strands 2d and 6d) are in light blue [68]. The second-binding site for PLC-β2 found in the N-terminal region of Gβ are in magenta [71]. The Gγ subunit is colored in dark grey.…”

“…In addition, it was found that the geranylgeranylated moiety attached to the C-terminal segment of the Gγ, which is proximal to the membrane and anchors Gβγ to lipids, binds to the PLC-β2 [69,70]. Moreso, Smrcka and colleagues, proposed that the 23-27 region of the N-terminal helix of the Gβ subunit was a second PLC-β binding site maintaining the enzyme outside the propeller in an inhibition state [71]. Thus, it appears that the top and the edge sides of the propeller is the region of Gβγ which binds to PLC-β and promotes activation.…”

“…In line with this, by swapping the PH domain of PLC-β2 into the remainder of PLC-δ1, we obtained a chimera that binds non-specifically to membranes like PLC-β2, and moreover, binds and is responsive to Gβγ subunits [72]. In two distinct studies, it was found that the N-terminal region of PLC-β3 encompassing the PH domain and a part of the EF-hands region binds to Gβγ [73] whereas it was reported that Gβγ helps the recruitment of a GFP-tagged PH-β1 on subcellular membranes [74] Additionally, an independent series of studies located a solvent-accessible segment of nine residues (574-583 segment) in the Y region that is able to bind to Gβ [71,[75][76][77]. This segment, although partially-conserved in PLC-δ1, could govern the oscillation of the PLC-β2 between two distinct parts of Gβ subunit to be distal or proximal to the membrane surface [71].…”

“…Comparing a model structure of PH-β2 with the known structure of PH-δ1 suggests that the β5/β6 loop, which is longer than in PH-δ1, (Figure 3) could act as a regulatory domain for activation by Gβγ [41]. A peptide corresponding to this loop (PHβ [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] ) activates the enzyme, and this activation is competitive with Gβγ activation. The structure of PLC-β2 reveals that the β5/β6 loop does not belong to the surface that is tightly packed with the EF-hands and the catalytic regions, or to the hydrophobic ridge interacting with Rac2.…”

“…The ability of the peptide corresponding to PHβ [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] to activate the enzyme was unexpected.…”