Regions in the G Protein Subunit Important for Interaction with Receptors and Effectors

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 Gi1 (range, 0.5–1.2 nM) and were most effective at coupling the Gi1 subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase Cisoform 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 Nand Cterminal regions of the subunit impart specificity to receptor and effector interactions. Among the best characterized signal transduction systems are the pathways used by receptors coupled to heterotrimeric G proteins. The G protein subunit plays an important role in these pathways. It is required for the interaction between the receptor and the subunit to initiate GDP/GTP exchange and also stabilizes the basal state of the receptor-GDPcomplex, which forms the high-affinity ligand binding conformation of receptors (Cabrera-Vera et al., 2003). Once released from the activated subunit, the subunit can regulate more than 20 effectors, including PLC, adenylyl cyclase, ion channels (Hildebrandt, 1997; Cabrera-Vera et al., 2003), PtdIns 3-kinase (Kerchner et al., 2004), and guanine nucleotide exchangers for small GTP binding proteins (Welch et al., 2002). The free dimer can also participate in regulatory events by binding to cytoplasmic proteins such as the -adrenergic receptor kinase or phosducin (Cabrera-Vera et al., 2003). It is noteworthy that the multiple isoforms of the dimer can differentially regulate effectors (Hildebrandt, 1997; Cabrera-Vera et al., 2003), making it necessary to understand the regions of these subunits that interact with