CONSTITUTIVELY ACTIVE MUTANTS OF THE ALPHA(1B)-ADRENERGIC RECEPTOR - ROLE OF HIGHLY CONSERVED POLAR AMINO-ACIDS IN RECEPTOR ACTIVATION

Site-directed mutagenesis and molecular dynamics simulations of the al pha(1B)-adrenergic receptor (AR) were combined to explore the potentia l molecular changes correlated with the transition from R (inactive st ate) to R (active state). Using molecular dynamics analysis we compar ed the structural/dynamic features of constitutively active mutants wi th those of the wild type and of an inactive alpha(1B)-AR to build a t heoretical model which defines the essential features of R and R. The results of site-directed mutagenesis are in striking agreement with t he predictions of the model supporting the following hypothesis. (i) T he equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY m otif. In fact, replacement of D142 with alanine confers high constitut ive activity to the alpha(1B)-AR. (ii) The shift of R143 of the DRY se quence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Di sruption of these intramolecular interactions by replacing N63 with al anine constitutively activates the alpha(1B)-AR. Our findings might pr ovide interesting generalities about the activation process of G prote in-coupled receptors.