Abstract
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.
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