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We have monitored agonist-induced
Volume 270,
Number 15,
Issue of April 14, pp. 8902-8909, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Adrenergic Receptor Subcellular
Distribution and Function
-adrenergic
receptor (
AR) redistribution by immunocytochemical
procedures in concert with functional measurements of agonist-elicited
[
H]inositol phosphate (InsP) production in human
embryonal kidney 293 cells stably expressing AR cDNA
(HEK293/
). Anti-peptide antibodies directed against
the carboxyl-terminal decapeptide of the
AR were
prepared and shown to react specifically with
AR on
immunoblots and in situ in HEK293/
transfectants. Treatment of HEK293/
cells with
norepinephrine (10 µ
M) results in a rapid (5-15 min)
and striking internalization of cell surface receptor as visualized by
confocal immunofluorescence microscopy. Receptor redistribution is
sustained in the presence of agonist, rapidly reversed upon agonist
removal, and prevented by the
antagonist prazosin.
Receptor internalizes to endosomes, as shown by colocalization with
transferrin receptor, an endosomal marker. Activation of protein kinase
C (PKC) with phorbol 12-myristate 13-acetate (50 n
M) causes
receptor endocytosis similar to agonist; agonist-induced
internalization is blocked by the PKC inhibitor staurosporine (0.5
µ
M). In parallel experiments, agonist-induced
[H]InsP production is abolished by phorbol
12-myristate 13-acetate but potentiated by staurosporine. Inhibition of
receptor internalization with hypertonic sucrose attenuates
agonist-induced [H]InsP formation; this effect is
reversed by concomitant inhibition of PKC with staurosporine. These
results suggest that PKC-dependent phosphorylation occurring as a
consequence of AR stimulation induces receptor
desensitization and internalization. Internalized receptor is
reactivated and continuously recycled to the cell surface during
agonist exposure.
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