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1B-Adrenergic Receptor
that Differentiates Two Steps in the Pathway of Receptor
Internalization
Department of Pharmacology, University of Nebraska Medical Center,
Omaha, Nebraska 68198-6260
An NP(X)nY motif is highly conserved among G
protein-coupled receptors and is similar to an NPXY motif involved in
receptor-mediated endocytosis for several non-G protein-coupled
receptors. We investigated the role of this motif in
1B-adrenergic receptor function and regulation. Y348A
1B-adrenergic receptors in which this sequence was
mutated from NPIIY to NPIIA were prepared by site-directed mutagenesis
and transfected into Chinese hamster ovary cells. Binding of the
antagonist prazosin to Y348A receptors was similar to that of wild-type
receptors, but affinity of the Y348A receptors for the agonist
epinephrine was increased by ~10-fold. Despite this increase in
agonist binding affinity, the Y348A mutation completely uncoupled the
receptors from stimulation of phosphoinositide hydrolysis and
mobilization of intracellular Ca2+. Exposure of cells
expressing Y348A receptors to the agonist epinephrine resulted in
receptor "sequestration," defined as a loss of cell surface
receptors accessible to radioligand in binding assays with intact cells
on ice, similar to that for the wild-type receptor. In contrast, Y348A
receptors did not undergo "endocytosis" into the light vesicle
fraction in sucrose density gradient centrifugation assays, as did the
wild-type receptor. These results (i) indicate an important role for
Tyr348 in coupling the 1B-adrenergic receptor to G
protein and subsequent effector activation, (ii) provide further
evidence that 1B-adrenergic receptor internalization can
be separated into a sequestration step and an endocytosis step, (iii)
indicate that effector activation and second messenger formation are
not required for the sequestration of these receptors but may be
involved in endocytosis, and (iv) provide a useful new tool for further
investigation of the nature of the subcellular compartments and the
molecular modifications involved in the multiple steps involved in
internalization of G protein-coupled receptors.
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