Molecular Basis for Interactions of G Protein βγ Subunits with Effectors

Abstract

Both the ␣ and ␤␥ subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. G␤␥ subunits can regulate a diverse array of effectors, including ion channels and enzymes. G␣ subunits bound to guanine diphosphate (G␣-GDP) inhibit signal transduction through G␤␥ subunits, suggesting a common interface on G␤␥ subunits for G␣ binding and effector interaction. The molecular basis for interaction of G␤␥ with effectors was characterized by mutational analysis of G␤ residues that make contact with G␣-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-␤2, and ␤-adrenergic receptor kinase revealed the G␤ residues required for activation of each effector and provides evidence for partially overlapping domains on G␤ for regulation of these effectors. This organization of interaction regions on G␤ for different effectors and G␣ explains why subunit dissociation is crucial for signal transmission through G␤␥ subunits.Upon receptor activation, G proteins dissociate into free G␣ and G␤␥ subunits that can activate various effectors (1). Effector proteins of the G␤␥ complex include phospholipases (2), adenylyl cyclases (3), ion channels (4), G protein-coupled receptor kinases (5) and phosphoinositide 3-kinases (6). Other potential G␤␥ effectors include dynamin I and the nonreceptor protein tyrosine kinases Btk and Tsk (7). GDP-bound G␣ subunits (G␣-GDP) can compete with G␤␥ effectors and deactivate G␤␥-dependent signaling, suggesting that G␤␥ may use a common binding surface for interaction with G␣ and with its diverse effectors. Two regions on G␤␥ that interact with G␣ have been defined by the crystal structures of heterotrimeric G␣␤␥ (8), the switch interface (G␤ residues 57, 59, 98, 99, 101, 117, 119, 143, 186, 228, and 332) and the NH 2 -terminal interface (G␤ residues 55, 78, 80 and 89). Each of these residues on retinal G␤ (G␤1) was substituted with alanine, and each G␤1 mutant was expressed with either G␥1 or G␥2, two isoforms of the G␥ subunit. All mutated G␤1␥1 dimers were folded properly, were post-translationally modified appropriately, and were expressed at similar amounts as in the wild type (9). The G␤␥ mutants were tested for their ability to assemble into heterotrimers with G␣, to be activated by rhodopsin, and to interact with G␤␥ downstream signaling partners: ␤-adrenergic receptor kinase (␤ARK), phospholipase C-␤2 (PLC-␤2), adenylyl cyclase 2 (AC2), muscarinic potassium channel (GIRK1/GIRK4), and the calcium channel ␣1B subunit (CC␣1B).To determine whether purified G␤1H 6 ␥1 mutants could form heterotrimers, we measured the ability of the G␤␥ mutants to facilitate pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation of transducin G␣-GDP (Gt␣) (10). All mutants could support some level of ADP ribosylation, although G␤ mutants Ile 80 3 Ala 80 (I80A), K89A, L117A, and W332A (11) showed reduced ability to form heterotrimers (Fig. 1A).Because G␤␥ ...

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“…Bottom -The sequence of the X/Y linkers in PLC-δ1 and PLC-β2. The region corresponding to the signal transfer regions [66,67] are represented in light green whereas the general PLC-β2 binding domain are in dark blue [65][66][67]. Additional regions found to be important for the Gβγ -activation of PLC-β2 (strands 2d and 6d) are in light blue [68].…”

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Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

“…The structure showed that Pdc bound the same face of G t βγ as G t α, with a great degree of overlap between the interaction surfaces. This face of Gβγ also constitutes a major interaction site for other Gβγ effectors, including G protein receptor kinase 2 (GRK2), phospholipase Cβ (PLCβ), adenylyl cyclase 2 (AC2), GIRK K + channels and N-type Ca 2+ channels [38]. In addition to the effector binding surface, Pdc also occluded the membrane binding surface of G t βγ and buried the C-terminal farnesyl group of G t γ in a cleft of G t β created by Pdc binding [36,37,39].…”

Gγ inDictyostelium: Its Role in Localization of Gβγ to the Membrane Is Required for Chemotaxis in Shallow Gradients

“…G␤s have seven WD repeats at their COOH-terminal that fold into ␤-propeller structures, containing the binding sites for G␣s and effectors (Ford et al, 1998;Li et al, 1998). The NH 2 -terminal regions of G␤s are variable and form tight coiled-coil structures with the diverse G␥s.…”