Palmitoylation of the alpha 2A-adrenergic receptor. Analysis of the sequence requirements for and the dynamic properties of alpha 2A-adrenergic receptor palmitoylation.

Previous studies have demonstrated that the alpha 2A-adrenergic receptor (alpha 2A AR) incorporates [3H]palmitate and that replacement of Cys442 by Ala or Ser eliminates detectable acylation without perturbing coupling to pertussis toxin-sensitive GTP-binding proteins (Kennedy, M. E., and Limbird, L. E. (1993) J. Biol. Chem. 268, 8003-8011) or, as shown here, without perturbing agonist-dependent receptor phosphorylation, in contrast to the consequences of eliminating beta 2-adrenergic receptor acylation. As a first step in revealing the functional role for this post-translational modification at the alpha 2A AR, we explored sequences in the alpha 2AAR which confer alpha 2AAR acylation and whether or not [3H]palmitoylation of the alpha 2AAR is dynamic. Deletion of the 7 terminal amino acids distal to Cys442 of the alpha 2AAR did not eliminate detectable [3H]palmitoylation of the alpha 2AAR, whereas truncation to Leu441 did, indicating both that Cys442 is the likely site for acylation and that sequences distal to Cys442 are not required for acylation at Cys442. Since mutation of sequences proximal to Cys442 altered overall receptor structure, based on markedly reduced detectable adrenergic receptor binding, proximal motifs required for palmitoylation of the alpha 2AAR could not be explored further. When the turnover of [35S]Met/Cys-labeled alpha 2AAR was compared with the turnover of the [3H]palmitate-labeled alpha 2AAR, it was of interest that agonist treatment accelerated the half-life of decay of the [3H]palmitate-labeled alpha 2AAR without detectable receptor down-regulation, providing evidence that the acylation of the alpha 2AAR may be a dynamic process.