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 (G1) was substituted with alanine, and each G1 mutant was expressed with either G␥1 or G␥2, two isoforms of the G␥ subunit. All mutated G1␥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 G1H 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␥ ...