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J. Biol. Chem., Vol. 275, Issue 41, 32037-32045, October 13, 2000
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From The Center for Experimental Therapeutics, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
Prostacyclin
(PGI2), the major product of cyclooxygenase in
macrovascular endothelium, mediates its biological effects through its
cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of
agonist-induced desensitization (Smyth, E. M., Hong Li, W., and
FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are
unclear. We have examined agonist-induced sequestration of hIP. Human
IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C
terminus to the green fluorescent protein (GFP), was coupled to
increased cAMP (EC50 = 0.39 ± 0.09 nM)
and inositol phosphate (EC50 = 86.6 ± 18.3 nM) generation when overexpressed in HEK 293 cells.
Iloprost-induced sequestration of HAhIP-GFP, followed in real time by
confocal microscopy, was partially colocalized to clathrin-coated
vesicles. Iloprost induced a time- and concentration-dependent
loss of cell surface HA, indicating receptor internalization, which was
prevented by inhibitors of clathrin-mediated trafficking and partially
reduced by cotransfection of cells with a dynamin dominant negative
mutant. Sequestration (EC50 = 27.6 ± 5.7 nM) was evident at those concentrations of iloprost that
induce PKC-dependent desensitization. Neither the PKC
inhibitor GF109203X nor mutation of Ser-328, the site for PKC
phosphorylation, altered receptor sequestration indicating that, unlike
desensitization, internalization is PKC-independent. Deletion of the C
terminus prevented iloprost-induced internalization, demonstrating the
critical nature of this region for sequestration. Internalization was
unaltered by cotransfection of cells with G protein-coupled receptor
kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant
negative mutant, indicating that GRKs and arrestins do not play a role
in hIP trafficking. The hIP is sequestered in response to agonist
activation via a PKC-independent pathway that is distinct from
desensitization. Trafficking is dependent on determinants located in
the C terminus, is GRK/arrestin-independent, and proceeds in part via a
dynamin-dependent clathrin-coated vesicular endocytotic
pathway although other dynamin-independent pathways may also be involved.
Internalization and Sequestration of the Human Prostacyclin
Receptor*
*
This work was supported by Grants HL-62250 and HL-57847 from
the National Institutes of Health and 9906209U (to E. M. S.) from the
American Heart Association.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Robinette Foundation Professor of Cardiovascular Medicine. To whom
correspondence should be addressed: Center for Experimental Therapeutics, University of Pennsylvania, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.:
215-898-1184; Fax: -215-573-9135; E-mail:
garret@spirit.gcrc.upenn.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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