Agonist Regulation of 
-Adrenergic Receptor Subcellular Distribution and Function (*)
- From the (1) Department of Pharmacology m/c 868, University of Illinois at Chicago, Chicago, Illinois 60612
- Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892
- § To whom correspondence should be addressed. Tel.: 312-996-5664; Fax: 312-996-1225.
Abstract
We have monitored agonist-induced α
-adrenergic receptor (α
AR) redistribution by immunocytochemical procedures in concert with functional measurements of agonist-elicited [3H]inositol phosphate (InsP) production in human embryonal kidney 293 cells stably expressing α
AR cDNA (HEK293/α
). Anti-peptide antibodies directed against the carboxyl-terminal decapeptide of the α
AR were prepared and shown to react specifically with α
AR on immunoblots and in situ in HEK293/α
transfectants. Treatment of HEK293/α
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 α1antagonist 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 [3H]InsP production is abolished by phorbol 12-myristate 13-acetate but potentiated by staurosporine. Inhibition of receptor
internalization with hypertonic sucrose attenuates agonist-induced [3H]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 α1AR stimulation induces receptor desensitization and internalization. Internalized receptor is reactivated and continuously
recycled to the cell surface during agonist exposure.
Footnotes
-
Pharmacology Research Associate of the National Institute of General Medical Sciences.
-
↵* This work was supported by National Institutes of Health Grant GM 41470 (to R. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- α1AR
-
α1-adrenergic receptor
- HEK cell
-
human embryonal kidney cell
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- NE
-
norepinephrine
- PMA
-
phorbol 12-myristate 13-acetate
- InsP
-
inositol phosphate
- Li-PSS
-
physiological salt solution supplemented with LiCl
- PKC
-
protein kinase C.
-
↵2D. Siwik and R. D. Brown, unpublished data.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
