A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated.
The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.
Pei
G
G
Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710.
Samama
P
P
Lohse
M
M
Wang
M
M
Codina
J
J
Lefkowitz
R J
RJ
D000318:Adrenergic beta-Agonists; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D020558:GTP Phosphohydrolases / Q000032:analysis; D005786:Gene Expression Regulation; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011869:Radioligand Assay; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; D007545:Isoproterenol