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J Biol Chem, Vol. 274, Issue 34, 23707-23718, August 20, 1999
From the Department of Biochemistry, Merville House, University
College Dublin, Belfield, Dublin 4, Ireland
The prostacyclin receptor (IP), a G
protein-coupled receptor, mediates the actions of the prostanoid
prostacyclin and its mimetics. IPs from a number of species each
contain identically conserved putative isoprenylation CAAX
motifs, each with the sequence CSLC. Metabolic labeling of human
embryonic kidney (HEK) 293 cells stably overexpressing the
hemagluttinin epitope-tagged IP in the presence of
[3H]mevalonolactone established that the mouse IP is
isoprenylated. Studies involving in vitro assays confirmed
that recombinant forms of the human and mouse IP are modified by carbon
15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed
mutagenesis of Cys414 to Ser414, within
the CAAX motif, abolished isoprenylation of
IPSSLC both in vitro and in transfected
cells. Scatchard analysis of the wild type (IP) and mutant
(IPSSLC) receptor confirmed that each receptor exhibited
high and low affinity binding sites for [3H]iloprost,
which were not influenced by receptor isoprenylation. Whereas stable
cell lines overexpressing IP generated significant agonist (iloprost
and cicaprost)-mediated increases in cAMP relative to nontransfected
cells, cAMP generation by IPSSLC cells was not
significantly different from the control, nontransfected HEK 293 cells.
Moreover, co-expression of the alpha (
) subunit of Gs generated
significant augmentations in cAMP by IP but not by IPSSLC
cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca2+]i in response
to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca2+]i by
IPSSLC was significantly impaired. Co-transfection of cells
with either Gq or G11 resulted in
significant augmentation of agonist-mediated [Ca2+]i mobilization by IP cells
but not by IPSSLC cells or by the control, HEK 293 cells.
In addition, inhibition of isoprenylation by lovastatin treatment
significantly reduced agonist-mediated cAMP generation by IP in
comparison to the nonisoprenylated 
2 adrenergic receptor
or nontreated cells. Hence, isoprenylation of IP does not influence
ligand binding but is required for efficient coupling to the effectors
adenylyl cyclase and phospholipase C.
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