Characterization of a Phospholipase C β2-Binding Site Near the Amino-terminal Coiled-coil of G Protein βγ Subunits

Abstract

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148 -7154), we showed that overlapping peptides, N20K (Asn 564 -Lys 583 ) and E20K (Glu 574 -Lys 593 ), from the catalytic domain of phospholipase C (PLC) ␤2 block G␤␥-dependent activation of PLC ␤2. The peptides could also be directly cross-linked to ␤␥ subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to G␤ 1 was inhibited by PLC ␤2 but not by ␣ i1 (GDP), indicating that the peptide-binding site on ␤ 1 represents a binding site for PLC ␤2 that does not overlap with the ␣ i1 -binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC ␤2 interaction with ␤ subunits. Each of the 14 cysteine residues in ␤ 1 were altered to alanine. The ability of the PLC ␤2-derived peptide to cross-link to each ␤␥ mutant was then analyzed to identify the reactive sulfhydryl moiety on the ␤ subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of ␤ 1 which is located in the amino-terminal coiled-coil region of ␤ 1 and identifies a PLC-binding site distinct from the ␣ subunit interaction site.Guanine nucleotide-binding proteins (G proteins) 1 are a large group of structurally similar proteins consisting of three subunits (␣, ␤, and ␥) that are central molecules coupling seven-transmembrane domain-spanning receptors to downstream effector molecules. Activation of G proteins begins with a ligand-induced conformational change of the receptor which catalyzes the release of GDP from the ␣ subunit in exchange for GTP (1, 2). In the GDP-bound heterotrimeric state, ␣(GDP)⅐␤␥, neither ␣(GDP) nor ␤␥ can regulate effector activity. Upon receptor-catalyzed G protein activation, the heterotrimer dissociates into free ␣(GTP) and free ␤␥ subunits. It is well understood that both ␣(GTP) and ␤␥ subunits can interact with a variety of downstream effector molecules including enzymes and ion channels. GTP is hydrolyzed to GDP, and reassociation of ␣(GDP) with ␤␥ results in deactivation of ␤␥-dependent signaling. Despite detailed knowledge of ␣-and ␤␥ subunit functions, the mechanism for how ␤␥ subunits activate its variety of effectors is not entirely understood.Effector-binding sites on the surface of ␤␥ are beginning to be mapped. The putative competition between ␣(GDP) and effectors for ␤␥ forms the premise for recent studies to map effectorbinding sites at the ␣ subunit-binding interface on ␤. The three-dimensional structure of the G protein heterotrimer reveals that the ␤ subunit is a ␤-propeller with seven "blades" and an amino-terminal ␣-helix (3, 4). The ␣ subunit binds to a portion of the top of the ␤-propeller and along side one of the blades of the propeller. Two groups have shown that alanine substitution of ␣-contacting residues on the top surface...

Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gβγ

“…The current data with AGS8 in both the yeast and mammalian systems along with the recent reports regarding direct regulation of G␤␥ (27,28) and diversity of G␤␥ binding partners (29,30) suggest that G␤␥ may possess additional protein binding sites for signal processing (28,31). Such G␤␥ binding partners may serve as effectors for mammalian G␤␥, influence receptor coupling to G␣␤␥ or localize G␤␥ within a larger signaling complex, where it can regulate specific signaling pathways independent of the classical heterotrimeric G␣␤␥.…”

WDR26 Functions as a Scaffolding Protein to Promote Gβγ-mediated Phospholipase C β2 (PLCβ2) Activation in Leukocytes

“…Previous work has found that G␤␥ contains multiple binding sites for PLC␤2. In addition to the G␣ contact surface, the N-terminal coiled coil domain and the outer surface of the G␤ are also involved in binding PLC␤2 (22,31). In the absence of other constraints, G␤␥ activates PLC␤2 primarily through the G␣ t contact region (26).…”

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

“…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.…”