Structural determinants required for receptor-mediated activation of G alpha(s)

Heptahelical transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have elucidated the contact surfaces between G protein subunits, but structural information is lacking on how the G protein {dollar}alpha{dollar} subunit associates with receptors and the mechanism by which receptors catalyze nucleotide exchange on the {dollar}alpha{dollar} subunit. The region of {dollar}rmalphasb{lcub}s{rcub},{dollar} that contributes to its coupling to the {dollar}beta{dollar}-adrenergic receptor was previously localized to the carboxyl terminal 40% of the molecule by means of an {dollar}rmalphasb{lcub}i2{rcub}/alphasb{lcub}s{rcub}{dollar} chimera. Using a scanning mutagenesis approach, five clusters of residues are identified within this region of {dollar}rmalphasb{lcub}s{rcub}{dollar} in which substitutions significantly impair its activation through the {dollar}beta{dollar}-adrenergic receptor. Based on the locations of these regions in the {dollar}alpha{dollar} subunit structure, they appear to play one of the following roles: direct interaction with the receptor (residues in the extreme carboxyl terminus) or intramolecular transmission of the receptor signal, both to the bound nucleotide (buried residues near the bound GDP) as well as to residues of the {dollar}alpha{dollar} subunit near the interface of the helical and GTPase domains (residues in two adjacent loops, including Switch III, which changes conformation upon binding GTP). The helical domain of {dollar}rmalphasb{lcub}s{rcub}{dollar} is implicated in its receptor-mediated activation, as a single substitution within the this domain (N167R) results in impaired receptor-mediated activation of {dollar}rmalphasb{lcub}s{rcub},{dollar} and the receptor couplings of an {dollar}rmalphasb{lcub}i2{rcub}/alphasb{lcub}s{rcub}{dollar} and an {dollar}rmalphasb{lcub}s{rcub}/alphasb{lcub}i2{rcub}/alphasb{lcub}s{rcub}{dollar} chimera, both of which effectively replace the helical domain of {dollar}rmalphasb{lcub}s{rcub}{dollar} with {dollar}rmalphasb{lcub}i2{rcub}{dollar} residues, are significantly reduced compared to {dollar}rmalphasb{lcub}s{rcub}.{dollar} Evidence is then presented to suggest that an intramolecular interaction between the GTPase domain and helical domain of {dollar}rmalphasb{lcub}s{rcub}{dollar} is required to facilitate proper receptor-catalyzed nucleotide exchange on the {dollar}alpha{dollar} subunit, via an opening of the interdomain space to allow release of the bound GDP.