Abstract
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.
Highlights
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 -adrenergic receptors (ARs)
Both the mouse and human 3ARs are S-palmitoylated at the canonical site within the C-terminal cytoplasmic tail, but the h3AR is S-palmitoylated at two additional Cys within the second and third intracellular loops
Summary
To examine S-palmitoylation of the 3AR, we used the acylRAC method (resin-assisted capture of fatty-acylated proteins) in which the thioester bond linking palmitate to Cys is cleaved with neutral hydroxylamine and the resultant free thiol is coupled to thiopropyl-Sepharose for pulldown and subsequent Western blot analysis [18]. Analysis by acyl-RAC of FLAG-tagged h3AR stably expressed in HEK293 cells demonstrated S-palmitoylation (Fig. 2A). To examine the role of S-palmitoylation on receptor-effector coupling of the 3AR, we transiently expressed, in HEK293 cells, WT or Cys-mutant h3AR or m3AR and, 24 h after transfection, assessed by ELISA cAMP production elicited by exposure for 5 min to the 3AR-specific synthetic ligand mirabegron [21]. We first assessed steady-state plasmamembrane abundance of WT versus Cys-mutant m3AR or h3AR in HEK293 cells transiently expressing FLAG-tagged receptor. To examine directly a possible role for S-palmitoylation in membrane-receptor stability, we labeled cells transiently expressing WT or Cys-mutant m3AR or h3AR with anti-FLAG Ab and, at subsequent intervals over 25 h, labeled unpermeabilized cells with fluorescent secondary Ab followed by FACS. S-palmitoylation of different sites within the h3AR regulates membrane-receptor abundance differentially: S-palmitoylation of Cys-153 and Cys-292 within the second and third intracellular loops, respectively, is required for efficient receptor processing/targeting, whereas S-palmitoylation of Cys-361/ 363 stabilizes the receptor at the plasma membrane
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