The diversity in the heterotrimeric G protein ␣, ␤, and ␥ subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five ␣ subunits (␣ i1 , ␣ i2 , ␣ o , ␣ s , and ␣ q ) to associate with three ␤ subunits (␤ 1 , ␤ 2 , and ␤ 5 ) dimerized to a ␥ 2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (␥ 2HF ). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the ␤ 1 ␥ 2HF dimer as it can be specifically eluted from an ␣ i1 -agarose column with GDP and AlF 4 ؊ , and purified ␤ 1 ␥ 2HF dimer stimulates type II adenylyl cyclase. The ␤ 1 ␥ 2HF and ␤ 2 ␥ 2HF dimers immobilized on an anti-FLAG affinity column bound all five ␣ subunits tested, whereas the ␤ 5 ␥ 2HF dimer bound only ␣ q . The ability of other ␣ subunits to compete with the ␣ q subunit for binding to the ␤ 5 ␥ 2HF dimer was tested. Addition of increasing amounts of purified, recombinant ␣ i1 to the ␣ q in a Sf9 cell extract did not decrease the amount of ␣ q bound to the ␤ 5 ␥ 2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF 4 ؊ and deactivated in the presence of equal amounts of the ␤ 1 ␥ 2HF or ␤ 5 ␥ 2HF dimers, only ␣ q bound to the ␤ 5 ␥ 2HF dimer. The ␣ q -␤ 5 ␥ 2HF interaction on the column was functional as GDP, and AlF 4 ؊ specifically eluted ␣ q from the column. These results indicate that although the ␤ 1 and ␤ 2 subunits interact with ␣ subunits from the ␣ i , ␣ s , and ␣ q families, the structurally divergent ␤ 5 subunit only interacts with ␣ q .All cells possess multiple signaling pathways that transmit signals from the hormones, autacoids, neurotransmitters, and growth factors in their environment. Complex biochemical mechanisms exist to discriminate, integrate, and modulate a cell's response to this constantly changing set of stimuli. One of the best characterized signal transduction systems is the pathway used by receptors coupled to heterotrimeric G proteins 1 (1, 2). Our current understanding of this signaling pathway shows it to be surprisingly complex with large families of proteins comprising the receptors, G proteins, and effectors (1, 3, 4) and important roles for both the ␣ and ␤␥ subunits of the heterotrimer in activating effectors (2, 4, 5). Moreover, some ligands activate multiple G proteins (1, 2, 6), and certain receptors activate the MAP kinase pathway (6, 7) and/or other tyrosine kinase signaling pathways (8). Thus, an important unsolved question in cell signaling is how a cell selects a response from the multiple possibilities available. Current evidence holds that specificity is determined at many levels. In addition to the tissue-specific expression of receptors, G proteins, or effectors (3), there are important protein-protein interactions involving the ␣ and ␤␥ subunits of the G protein heterotrimer that determine specificity. For example, the ␣ t subunit couples selectively to rhodopsin and the ␣ s subunit ...

G␤␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␤␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the ␤ 1 subunit, G␤␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-␤, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C-␤ isoform and inhibiting type I adenyl cyclase. In contrast, ␤␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.

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