Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β2-adrenergic receptor

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the β<inf>2</inf>-adrenergic receptor (β<inf>2</inf>-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315^7.42 and Ser319^7.46, on H7 for structure-function analysis. Replacing Ser319^7.46 with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~ 15-20% agonist-independent activity. Replacement of Ser319^7.46 with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315^7.42 and Ser319^7.46 are stabilizing β<inf>2</inf>-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315^7.42 with Trp286 ^6.48 and hydrogen bonding interactions of Ser319^7.46 with amino acids on H1-H2-H7 and with structural water. © 2011 Elsevier B.V.