Interplay between negative and positive design elements in Gα helical domains of G proteins determines interaction specificity toward RGS2.

Regulators of G protein signaling (RGS) proteins inactivate Gα subunits, thereby controlling G protein-coupled signaling networks. Among all RGS proteins, RGS2 is unique in interacting only with the Gαq but not with the Gαi subfamily. Previous studies suggested that this specificity is determined by the RGS domain and, in particular, by three RGS2-specific residues that lead to a unique mode of interaction with Gαq This interaction was further proposed to act through contacts with the Gα GTPase domain. Here, we combined energy calculations and GTPase activity measurements to determine which Gα residues dictate specificity toward RGS2. We identified putative specificity-determining residues in the Gα helical domain, which among G proteins is found only in Gα subunits. Replacing these helical domain residues in Gαi with their Gαq counterparts resulted in a dramatic specificity switch toward RGS2. We further show that Gα-RGS2 specificity is set by Gαi residues that perturb interactions with RGS2, and by Gαq residues that enhance these interactions. These results show, for the first time, that the Gα helical domain is central to dictating specificity toward RGS2, suggesting that this domain plays a general role in governing Gα-RGS specificity. Our insights provide new options for manipulating RGS-G protein interactions in vivo, for better understanding of their 'wiring' into signaling networks, and for devising novel drugs targeting such interactions.

"Disruptor" residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits.

Understanding the molecular basis of interaction specificity between RGS (regulator of G protein signaling) proteins and heterotrimeric (αßγ) G proteins would enable the manipulation of RGS-G protein interactions, explore their functions, and effectively target them therapeutically. RGS proteins are classified into four subfamilies (R4, R7, RZ, and R12) and function as negative regulators of G protein signaling by inactivating Gα subunits. We found that the R12 subfamily members RGS10 and RGS14 had lower activity than most R4 subfamily members toward the Gi subfamily member Gαo Using structure-based energy calculations with multiple Gα-RGS complexes, we identified R12-specific residues in positions that are predicted to determine the divergent activity of this subfamily. This analysis predicted that these residues, which we call "disruptor residues," interact with the Gα helical domain. We engineered the R12 disruptor residues into the RGS domains of the high-activity R4 subfamily and found that these altered proteins exhibited reduced activity toward Gαo Reciprocally, replacing the putative disruptor residues in RGS18 (a member of the R4 subfamily that exhibited low activity toward Gαo) with the corresponding residues from a high-activity R4 subfamily RGS protein increased its activity toward Gαo Furthermore, the high activity of the R4 subfamily toward Gαo was independent of the residues in the homologous positions to the R12 subfamily and RGS18 disruptor residues. Thus, our results suggest that the identified RGS disruptor residues function as negative design elements that attenuate RGS activity for specific Gα proteins.