Distinct Sites on G Protein βγ Subunits Regulate Different Effector Functions

Abstract

G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of G␤␥ that are crucial for G protein-coupled inward rectifier K ؉ (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K ؉ currents. Each of the three G␤ 1 mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K ؉ channels. With N-type calcium channels, G␤␥ wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved G␤␥ inhibition of Ca 2؉ currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant G␤␥ subunits activated phospholipase C ␤ 2 with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the G␤␥ regulation of a specific effector function without affecting other G␤␥-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on G␤ 1 that regulates several effectors. These data show that distinct domains within G␤␥ subserve specific functional roles.G protein ␤␥ subunits interact with several effectors including G protein-gated K ϩ channels, N-type calcium channels and phospholipase C␤ (1). Several reports have investigated the molecular sites of interactions between G␤␥ and their effectors, but the exact molecular mechanism by which G␤␥ alters effector function remains unclear. In one case, the co-crystal structure of the G␤ 1 ␥ 2 and phosducin has been determined (2). This structure points to multiple interactions between the two proteins concentrated in at least two distinct domains. Other studies using indirect approaches including mutagenesis have corroborated this design for G␤␥ interaction with its effectors (2-5). Most of these studies have identified multiple sites on G␤␥ as well as on each effector, where such interactions may occur.We have recently reported multiple sites of interactions between GIRK channels and G␤␥ subunits based on chimeric analysis and mutagenesis studies (6 -8). First, we showed that there are at least three interaction sites on the GIRK channel that are crucial both for binding to G␤␥ and for transducing the G␤␥ effects on channel activity. Recently, we also identified eight residues on G␤ 1 that are important in functional interactions with GIRK 1 channels (8). These amino acids reside on distinct domains of the G protein as assessed from the known crystal structure (9, 10), consistent with the notion of multiple interactions between the two proteins.Although several interaction points between G␤␥ and ef...

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G protein inhibition of CaV2 calcium channels

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Direct G Protein Modulation of Cav2 Calcium Channels

“…Left image: 1, D186; 2, T143; 3, N119; 4, M101; 5, K78; 6, I80; 7, L55; 8, W332. Right image: 1, Y111; 2, D153; 3, S189; 4, S67 (Ford et al, 1998;Mirshahi et al, 2002a;Doering et al, 2004;Tedford et al, 2005). Residues indicated in yellow: A, N35 to N36; these mediate disinhibitory cross-talk with the PKC pathway via interactions with residue T422 of the N-type channel.…”