Abstract
Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes
a rapid desensitization of the receptor-stimulated adenylyl cyclase
response. Phosphorylation of the beta 2AR by several distinct kinases
plays an important role in this desensitization phenomenon. In this
study, we have utilized purified hamster lung beta 2AR and stimulatory
guanine nucleotide binding regulatory protein (Gs), reconstituted
in phospholipid vesicles, to investigate the molecular properties
of this desensitization response. Purified hamster beta 2AR was phosphorylated
by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or
beta AR kinase (beta ARK), and receptor function was determined by
measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity.
At physiological concentrations of Mg2+ (less than 1 mM), receptor
phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC),
and 30% (beta ARK). The desensitizing effect of phosphorylation was,
however, greatly diminished when assays were performed at concentrations
of Mg2+ sufficient to promote receptor-independent activation of
Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction
component involved in the attenuation of rhodopsin function, did
not enhance the uncoupling effect of beta ARK phosphorylation of
beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations
of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation
of beta ARK-mediated desensitization was observed upon arrestin addition.
At a free Mg2+ concentration of 5 mM, arrestin did not potentiate
the inhibition of receptor function observed on PKA or PKC phosphorylation.
These results suggest that distinct pathways of desensitization exist
for the receptor phosphorylated either by PKA or PKC or alternatively
by beta ARK.
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