THE CONSERVED 7-TRANSMEMBRANE SEQUENCE NP(X)(2,3)Y OF THE G-PROTEIN-COUPLED RECEPTOR SUPERFAMILY REGULATES MULTIPLE PROPERTIES OF THE BETA(2)-ADRENERGIC RECEPTOR

The beta(2)-adrenergic receptor (beta(2)AR) is a member of a large superfamily of seven transmembrane domain, G-protein-coupled receptors. Within the putative seventh transmembrane domain of the B(2)AR is a sequence of amino acids, NPLIY, which is conserved with minor variations in all members of the superfamily. Previously it was demonstrated that mutation of tyrosine residue 326 to an alanine abolished agonist promoted sequestration of this mutant without affecting its ability to maximally stimulate adenylyl cyclase in membranes [Barak, L. S., Tiberi, M., Freedman, N. J., Kwatra, M. M., Lefkowitz, R. J., & Caron, M. J. (1994) J. Biol. Chem. 269, 2790-2795]. In the present study we characterized the NPLIY amino acid sequence in an attempt to determine how it can affect the agonist-mediated sequestration of the beta(2)AR and to test whether it is a functional motif. We find that point mutations of the most conserved amino acids, N, P, and Y, in this sequence affect several other receptor properties in addition to sequestration. Mutation of asparagine 322 to an alanine resulted in complete uncoupling of the receptor, loss of high-affinity agonist binding, and abolition of receptor sequestration, down-regulation, and phosphorylation. In contrast, a conservative mutation of this residue to an aspartic acid (as found in the thrombin receptor) resulted in an improvement of G-protein coupling without adversely affecting other receptor properties. Substitution of proline residue 323 with an alanine residue resulted in a receptor with mild deficits in sequestration and coupling, a reduced agonist-mediated phosphorylation, and no change in down-regulation. A mutant receptor with tyrosine;residue 326 changed to a phenylalanine sequestered at 25% the rate of wild type receptor and was also phosphorylated less well than wild type receptor in response to agonist. In contrast, the alanine mutant of tyrosine 326 does not sequester and is weakly phosphorylated in response to agonist, and it activates adenylyl cyclase less well than wild type-receptor in whole-cell determinations. An insertion mutant, (IY)-I-325 --> (325)IAY, was inactive. These data suggest that the NPLIY sequence of the beta(2)AR functions as a motif that may represent a critical determinant for maintaining the normal conformation of the receptor but does not function as a specific sequestration recognition motif.