Differential S-palmitoylation of the human and rodent β₃-adrenergic receptors

With few reported exceptions, G protein-coupled receptors (GPCRs) are modified by Cys palmitoylation (S-palmitoylation). In multiple GPCRs, S-palmitoylation targets a canonical site within the C-terminal cytoplasmic tail adjacent to the C terminus of the seventh transmembrane domain, but modification of additional sites is exemplified by the β-adrenergic receptors (βARs). The β₁AR is S-palmitoylated at a second, more distal site within the C-terminal tail, and the β₂AR is modified at a second site within the third intracellular loop, neither of which is conserved in other βAR isoforms. The functional roles of S-palmitoylation of disparate sites are incompletely characterized for any GPCR family. Here, we describe S-palmitoylation of the β₃AR. We compared mouse and human β₃ARs and found that both were S-palmitoylated at the canonical site within the C-terminal tail, Cys-358 and Cys-361/363 in mouse and human β₃ARs, respectively. Surprisingly, the human β₃AR was S-palmitoylated at two additional sites, Cys-153 and Cys-292 within the second and third intracellular loops, respectively. Cys-153 is apparently unique to the human β₃AR, and Cys-292 is conserved primarily in primates. Mutational substitution of C-tail Cys in human but not mouse β₃ARs resulted in diminished ligand-induced cAMP production. Substitution of Cys-153, Cys-292, or Cys-361/363 within the human β₃AR diminished membrane-receptor abundance, but only Cys-361/363 substitution diminished membrane-receptor half-life. Thus, S-palmitoylation of different sites differentially regulates the human β₃AR, and differential S-palmitoylation distinguishes human and rodent β₃ARs, potentially contributing to species-specific differences in the clinical efficacy of β₃AR-directed pharmacological approaches to disease.