(Received for publication, February 26, 1997, and in revised form, May 30, 1997)
From the Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
The receptor for platelet-activating factor (PAF)
is a member of the G-protein-coupled receptor family. To study the
structural elements and mechanisms involved in the internalization of
human PAF receptor (hPAFR), we used the following mutants: a truncated mutant in the C-terminal tail of the receptor (Cys317
Stop) and mutations in the (D/N)P(X)2,3Y
motif (Asp289
Asn,Ala and Tyr293
Phe,Ala). Chinese hamster ovary cells expressing the Cys317
Stop mutant exhibited a marked reduction in their capacity to
internalize PAF, suggesting the existence of determinants important for
endocytosis in the last 26 amino acids of the cytoplasmic tail.
Substitution of Asp289 to alanine abolished both
internalization and G-protein coupling, whereas substitution of
Tyr293 to alanine abolished coupling but not
internalization. Inhibition or activation of protein kinase C did not
significantly affect the internalization process. Receptor
sequestration and ligand uptake was, at least in part, blocked by
concanavalin A and blockers of endocytosis mediated by clathrin-coated
pits. Our data suggest that the internalization of a G-protein-coupled
receptor and coupling to a G-protein can be two independent events.
Moreover, the C terminus tail of hPAFR, but not the putative
internalization motifs, may be involved in the internalization of
hPAFR.
Platelet-activating factor (PAF)1 is a potent phospholipid mediator that produces a wide range of biological responses through activation of a specific receptor on target cell surface (1, 2). The PAF receptor is part of the large family of seven transmembrane domain receptors (3-7). These receptors, when exposed to an agonist, activate a heterotrimeric G-protein that in turn activates and regulates events leading to cellular responses. Following repeated or prolonged agonist stimulation, their signal transduction potential becomes modulated and attenuated, a phenomenon termed desensitization (8). Three distinct events participate in the process of desensitization as follows: the functional uncoupling of the receptor from the G-protein, internalization or sequestration of the receptor from the cell surface toward the intracellular compartments, and finally, the down-regulation of receptor numbers, involving the degradation of the protein in lysosomes (9). The role of phosphorylation in receptor desensitization has been widely studied. It seems clear, at this time, that phosphorylation by second messenger kinases (PKC, PKA, PKG) and the GRKs (G-protein-coupled receptor kinases) helps to maintain the receptors uncoupled from the G-protein (10-13). However, the role of phosphorylation in receptor sequestration and down-regulation is less defined and seems to vary from receptor to receptor (14, 15).
Receptor internalization permits ligand entry and seems to participate
in resensitization as well as desensitization (16-20). The
internalization process can be mediated by clathrin-coated vesicles for
some receptors (17, 21), but not for others (22, 23), and a
phosphorylation step may or may not be essential (24). The structural
elements involved in this phenomenon can be found in the intracellular
loops (25-27) as well as the C-terminal tail (28-32) of the
individual receptors. No consensus motif has as yet been found,
although both positive (28, 30-32) and negative (29, 33) regulatory
elements have been identified for some receptors. The motif
(D/N)P(X)2,3Y is found at the end of the 7th
transmembrane domain of most receptors of this family and seems
involved in the internalization process of some (17, 34) but not all
(35-37) receptors. In addition, the substitution of residues in this
motif was found to affect other functions of the receptor, such as
G-protein coupling, affinity for the ligand (37, 38), and even the
phosphorylation level of the receptor. These results suggest a global
role for this region in the conservation of adequate receptor
conformation. It has also been shown that the medium chains
(µ1 and µ2) of clathrin-associated protein
complexes AP-1 and AP-2 can specifically interact with a tyrosine-based signal YXX (
is an amino acid with a bulky hydrophobic
side chain) motif (39). Both these motifs are found in the sequence DPVIYCFL present in the hPAFR.
It has been shown that the PAFR undergoes a ligand-specific,
temperature-dependent internalization in transfected cells
(40). The aim of our study was to identify the structural elements and mechanisms involved in the internalization of the hPAFR. The role of
the two motifs (D/N)P(X)2,3Y and
YXX was evaluated with the mutant receptors
Asp289
Ala,Asn (41) and Tyr293
Ala,Phe
(this study). The presence of regulatory elements in the cytoplasmic
tail was verified with the mutant Cys317
Stop that does
not contain the last 26 amino acids of the C-terminal tail. The
involvement of clathrin-coated vesicles in hPAFR internalization was
addressed with internalization blockers. Finally, to determine whether
signalization was an important step in the initiation or regulation of
the hPAFR internalization process, certain inhibitors and an activator
of PKC, in addition to uncoupled mutants of the receptor, D63N (43),
A230E (44), D289A (41), and Y293A (this study), were used.
Reagents were obtained from the following sources: oligonucleotides were synthesized at Life Technologies, Inc., Pwo polymerase was from Boehringer Mannheim, restriction endonucleases and T4 DNA ligase were from Promega and Pharmacia Biotech Inc., bovine serum albumin, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, pertussis toxin, phenylarsine oxide, and concanavalin A were from Sigma, staurosporine and calphostin C were from BIOMOL Research Laboratories, Inc., NH4Cl and sucrose were from Fisher, lipid-free bovine serum albumin and hexadecyl-PAF were from Calbiochem, AG1-X8 resin (in formate form, 100-200 mesh) was from Bio-Rad, cell culture media and LipofectAMINE were from Life Technologies, Inc., [3H]hexadecyl-PAF and myo-[2-3H]inositol were from Amersham Corp., and 3H-WEB2086 was purchased from NEN Life Science Products. The pJ3M expression vector (45) and the clone Kp132 containing the hPAFR cDNA (40) were kindly provided by Dr. J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA) and Dr. Richard Ye (The Scripps Research Institute, La Jolla, CA), respectively.
Construction of Epitope-tagged PAF Receptor cDNA and MutantsA tagged hPAFR cDNA was generated by polymerase chain
reaction (46) from Kp132 using the oligonucleotide
5-CCACATGACTCCTCCCACATG-3
and the M13 sequencing primer
(5
-GTAAAACGACGGCCAGT-3
). The resulting fragment was then digested
with Acc65I and subcloned into the Eco1CR1-Acc65I sites of the pJ3M vector. In this
construction, the N-terminal initiator methionine was replaced by the
peptide sequence MEQKLISEEDLSRGSPG, resulting in a c-myc
epitope-tagged PAF receptor coding sequence. Mutated receptors were
constructed by polymerase chain reaction using Kp132 as template.
Tyr293
Phe and Tyr293
Ala substitutions
were created using the oligonucleotides 5
-TGTTATCTTCTGTTTCC-3
and 5
-CCTGTTATCGCCTGTTTCC-3
with their reverse complements, respectively. The point mutation was then introduced in the
epitope-tagged receptor coding sequence using BstEII and
Acc65I restriction enzymes. Truncated forms,
Cys317
Stop and Lys298
Stop, of the
receptor were also constructed. To construct these mutants the
polymerase chain reaction product generated with the oligonucleotides
5
-CCACATGACTCCTCCCACATG-3
and 5
-GCCCGGGATCATTTCCGG-3
(Cys317
Stop) or 5
-CCAATTCTAGGTGAGGAAAC-3
(Lys298
Stop) were subcloned in the site
Eco1CR1 of pJ3M vector. Mutations and the integrity of the
coding sequence were confirmed by dideoxy sequencing (University of
Alberta, Alberta, Canada).
COS-7 and CHO cells were grown in Dulbecco's modified Eagle's medium high glucose and Dulbecco's modified Eagle's medium F12, respectively, supplemented with 10% fetal bovine serum. Cells were plated in 30-mm dishes (2.0 × 105 COS-7 cells/dish or 3.0 × 105 CHO cells/dish), transiently transfected with the constructions encoding the WT and the mutant receptors using 4 µl of LipofectAMINE and 1 µg of DNA per dish and harvested 48 h after transfection. For studies of biphasic isotherms, 100-mm dishes with 3 × 106 CHO cells/dish were transfected with constructions encoding the WT and Y293A mutant using 30 µl of LipofectAMINE and 4 µg of DNA. Stably transfected CHO cells (44) were grown in Dulbecco's modified Eagle's medium F12 containing 400 µg/ml G418.
Radioligand Binding AssayCompetition binding curves were done on COS-7 cells expressing the WT and mutant receptor species. Cells were harvested and washed twice in Hepes-Tyrode's buffer (140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mM NaHCO3, 5.6 mM D-glucose, 0.49 mM MgCl2, 0.37 mM NaH2PO4, 25 mM Hepes, pH 7.4) containing 0.1% (w/v) bovine serum albumin (BSA) (47). Binding reactions were carried out on 5 × 104 cells in a total volume of 0.25 ml in the same buffer with 10 nM 3H-WEB2086 and increasing concentrations of nonradioactive WEB2086 or PAF for 90 min at 25 °C. Reactions were stopped by centrifugation. The cell-associated radioactivity was measured by liquid scintillation.
Inositol Phosphate DeterminationCOS-7 cells were transfected as described above with the WT or mutant receptors and labeled the following day for 18-24 h with myo-[3H]inositol at 5 µCi/ml in Dulbecco's modified Eagle's medium (high glucose, without inositol). After labeling, cells were washed once in phosphate-buffered saline (PBS), pretreated or not with the indicated inhibitors (concanavalin A (0.25 mg/ml, 20 min), sucrose (0.45 M, 20 min), NH4Cl (10 mM, 10 min), phenylarsine oxide (80 µM, 5 min with 30-min rest)) and preincubated 5 min in PBS at 37 °C. At the end of this preincubation period, the PBS was removed, and cells were incubated in pre-warmed Dulbecco's modified Eagle's medium (high glucose, without inositol) containing 0.1% BSA and 20 mM LiCl for 5 min. Cells were then stimulated for 30 s with indicated concentrations of PAF. The reactions were terminated with the addition of perchloric acid followed by a 30-min incubation on ice. Inositol phosphates were extracted (48) and separated on Dowex AG1-X8 columns (49). Total labeled inositol phosphates were then counted by liquid scintillation.
Ligand InternalizationThe evaluation of ligand
internalization kinetics was done on CHO cells transiently transfected
in 12-well dishes with constructions encoding mutant and WT receptors.
48 h after transfection, cells were pretreated or not with the
indicated inhibitors (pertussis toxin (150 ng/ml, 20 h),
concanavalin A (0.25 mg/ml, 20 min), sucrose (0.45 M, 20 min), NH4Cl (10 mM, 10 min), phenylarsine oxide
(80 µM, 5 min with 30 min rest), calphostin C (2 µM, 20 min), staurosporine (3 µM, 20 min),
and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 µM, 20 min]) or 4-PMA (inactive) or 4
-PMA (active)
(80 nM, 30 min)) and then incubated at 37 °C with 2 nM [3H]hexadecyl-PAF in a buffer containing
150 mM choline chloride, 10 mM Tris-HCl, pH
7.5, 10 mM MgCl2, and 0.25% lipid-free BSA (40) for 5, 10, 20, 30, 45, or 100 min. After the incubation period,
cells were washed two times with 1 ml of the same buffer but containing
2% BSA. Cells were then lysed in 0.1 N NaOH, and internalized radioactivity was evaluated by liquid scintillation.
Stably transfected CHO cells were grown on coverslips (25 mm). The cells were incubated in the presence or absence of sucrose and/or PAF (0.5 µM) for 20 min and fixed with 4% paraformaldehyde (15 min at RT). The coverslips were then placed in 0.1% Triton (20 min RT) and then sequentially incubated with 5% milk (30 min RT) and 0.1% glycine (60 min RT). The cells were then incubated with anti-PAFR antibodies (polyclonal, directed at the C-terminal tail; Cayman Chemical, Ann Arbor, MI) followed with rhodamine-conjugated goat anti-rabbit antibodies (Bio/Can Scientific, Mississauga, Ontario). The cells were then analyzed on a Molecular Dynamics (Sunnyvale, CA) Multi-Probe 2001 confocal argon laser scanning system equipped with a Nikon Diaphot epifluorescence inverted microscope. Scanned images were transferred onto a Silicon Graphics Indy 4000 workstation equipped with Molecular Dynamics' Imagespace analysis software.
The
mutants used in the present report are illustrated in Fig.
1. The deletion mutants,
Cys317 Stop and Lys298
Stop, were made
to analyze the involvement of the cytoplasmic tail in the
internalization of the receptor. The tyrosine 293 putatively involved
in the motifs (N/D)P(X)2,3Y and
YXX
was substituted with phenylalanine and alanine. The
role of receptor-mediated signal transduction in internalization was
studied with the help of G-protein-uncoupled mutants that were
distributed in distinct areas of the receptor (D63N, A230E, D289A, and
Y293A).
The binding characteristics of the WT receptor and the mutants,
Cys317 Stop, Y293A, and Y293F, were examined in
transiently transfected COS-7 cells using the agonist (PAF) and an
antagonist (WEB2086) (Fig. 2). The
Lys298
Stop mutant was not illustrated as it did not
bind either of the ligands, despite comparable cell-surface expression
to the WT construct, as examined by cytofluorimetry with an
anti-c-myc antibody (results not shown). No significant
difference was found in the affinities for WEB2086 (Fig. 2A)
or PAF (Fig. 2B) between the different mutants and the WT
receptor. Similar levels of expression were also obtained in COS-7 as
well as in CHO cells (WT, 962,310 ± 163,592; Y293A, 961,945 ± 146,532; Y293F, 857,332 ± 155,432 receptors/CHO cell) except
for Cys317
Stop (342,578 ± 68,515 receptors/CHO
cell) which was expressed at a lower level.
To further characterize these receptors, the signaling response of the
mutant (Cys317 Stop, Y293A, and Y293F) and WT receptors
was measured by IP accumulation (Fig.
3A). In COS-7 cells,
hPAFR-induced IP production would be mediated by PLC
1 activation via
Gq/11 as the IP production is not blocked by pertussis
toxin or tyrosine kinase inhibitors (44). Transiently transfected COS-7
cells were exposed for 30 s to graded concentrations of PAF, and
total IP accumulation was measured. As shown, the Cys317
Stop deletion of the cytoplasmic tail did not impair the response of the receptor. Since nearly half the potential phosphorylation sites
of the cytoplasmic tail were eliminated by this deletion, the slightly
augmented response of this mutant could reflect the role played by
these residues in controlling the response to PAF. On the other hand,
the Y293A mutant did not generate an IP response, except at very high
concentrations of PAF (1 µM). In contrast, the
substitution of Y293F had no significant effect on the response of the
receptor.
When conditions of transfection are such that low levels of hPAFR receptors are expressed (44), receptors can be observed in both a G-protein-coupled state (high affinity) and uncoupled state (low affinity). As shown in Fig. 3B, under these conditions of receptor expression, the mutant Y293A appears to exist only in its uncoupled, low affinity state, when compared with the WT, which might explain its inability to activate PLC after PAF stimulation.
Characterization of the Internalization PathwaysThe effects
of different inhibitors of internalization on receptor sequestration
and endocytosis of 3H-PAF are illustrated in Fig.
4. After a 20-min stimulation of stably
transfected hPAFR-CHO cells with 0.5 µM PAF,
sequestration was studied by confocal microscopy with an antibody
raised against a peptide sequence of the C-terminal portion of the
hPAFR (Fig. 4A). Cells were pretreated with medium
(a and c) or with hyperosmolar concentration of
sucrose (b and d) to block clathrin-mediated internalization before stimulation with PAF (c and
d). PAF induced the translocation of receptors toward the
intracellular compartment (c), whereas sucrose blocked the
process of hPAFR sequestration (d).
Effect of inhibitors of internalization on hPAFR sequestration (A), 3H-PAF endocytosis (B), and response (C). CHO cells stably transfected with the WT receptor were used for the following studies. A, cells were incubated with medium (a and c) or with sucrose (b and d) and then exposed (c and d) to 0.5 µM PAF, 20 min at 37 °C before fixation and permeabilization; sequestration of hPAFR was then revealed in confocal microscopy with an anti-PAFR antibody and a rhodamine-conjugated goat anti-rabbit antibody, as described under "Experimental Procedures." B, cells were incubated with 2 nM 3H-PAF for 30 min at 37 °C and washed in the presence of 2% BSA to remove the extracellular PAF. 3H-PAF internalization was determined after subtraction of nonspecific values that were obtained in the presence of 5 µM PAF. The effect of inhibitors on IP production was examined in COS-7 cell transiently transfected with the WT receptor (C). Cells were stimulated with PAF 100 nM for 30 s after a pretreatment with medium (control) or the inhibitors. IP quantification was as described under "Experimental Procedures." The results are representative of three independent experiments, each done in duplicate.
In addition, sucrose as well as other inhibitors of clathrin-mediated endocytosis significantly blocked the internalization of 3H-PAF (Fig. 4B), indicating that receptor-specific endocytosis of PAF is mediated via clathrin-coated vesicles. Fig. 4C shows that inhibition of PAFR endocytosis was not a nonspecific effect of the inhibitors on ligand binding or receptor signal transduction. The level of IP accumulation was measured after PAF stimulation in the presence of the inhibitors, which, except for phenylarsine oxide, did not significantly inhibit PAFR response. In fact, the apparent decrease in PAF-induced response in cells pretreated with phenylarsine oxide was due to enhanced basal levels of IP production.
The Role of Signal Transduction in hPAFR InternalizationThe
different mutants were compared with the WT receptor for their capacity
to internalize 3H-PAF (Fig.
5). The Lys298 Stop
mutant was used as a control for non-receptor-mediated 3H-PAF internalization since it did not detectably bind
PAF. The results indicate that the majority of mutants that are
uncoupled from G-proteins (Fig. 5A) were impaired in their
capacity to internalize PAF, except for the Y293A mutant. This mutant
indicated that receptor signalization does not participate in
ligand-mediated internalization of hPAFR. Although PAF-induced
mitogen-activated protein kinase activation in guinea pig
PAFR-transfected CHO cells was reported to be pertussis toxin-sensitive
(47), internalization of human PAFR in stably transfected CHO cells was
not affected by pertussis toxin treatment (data not illustrated). In
contrast, the Cys317
Stop mutant was impaired in its
capacity to mediate PAF internalization (Fig. 5B), despite
its efficient signal transduction, indicating that the last 26 amino
acids of the C-terminal tail could be important for internalization. As
Cys317
Stop is expressed less than the WT, we expressed
the WT at different levels and compared the internalization. When the
WT is expressed at 30% maximal level (comparable to Cys317
Stop expression), its internalization is only 25% less than maximal WT internalization, whereas Cys317
Stop
internalizes 90% less than the WT. In addition, our results indicate
(Fig. 5B) that the substitution of the residues
Asp289 or Tyr293, within the putative
internalization motifs (N/D)P(X)2,3Y and YXX
, by conserved residues (Asn and Phe, respectively)
partially decreased but did not abolish the potential for
internalization.
Phosphorylation by PKC does not appear to be involved in the initiation
or control of PAFR internalization. Modulation of PKC activity by
inhibitors (calphostin C, staurosporine, and H7) or an activator, in
its active 4-PMA or inactive 4
-PMA form, did not affect
PAF-induced internalization (Fig. 6).
In agreement with data obtained by Takano et al. (50),
with a truncated guinea pig PAF receptor, our data indicate that the
last 26 amino acids of the C-terminal end of the hPAFR are not required
for functional coupling to a G-protein, as removal of this region
yielded a receptor that was as efficient as the WT in mediating
phospholipid hydrolysis. In contrast, as observed with our
Lys298 Stop mutant, a larger deletion in the
cytoplasmic tail can confer a loss of binding capacity for both PAF and
WEB2086. This loss of effector function could be the consequence of a
structural change in the 7th transmembrane domain, as substitution of
Asp285
Ile, in this domain, leads to a similar
phenotype (41). Our data indicate, however, that the intracellular
C-terminal tail of the hPAFR may be involved in endocytosis of the
receptor-ligand complex, as internalization of 3H-PAF was
dramatically decreased, although not completely suppressed, in the
Cys317
Stop mutant. These results indicate either that
other parts of the receptor might also be involved in controlling the
internalization process or that 3H-PAF could be partially
internalized by constitutive endocytosis; a similar deletion in the
thyrotropin-releasing hormone receptor led to a constitutively
internalized and recycled receptor (51). The role of different
intracellular domains of G-protein-coupled receptors in triggering
internalization has been demonstrated in several studies using
mutagenesis. Some segments in the intracellular loops (25-27) as well
as in the C-terminal domain of the yeast pheromone
-factor,
thyrotropin, gastrin, parathyroid hormone, and angiotensin receptors
were found to be specifically involved in the sequestration process (5,
6, 8, 51), whereas deletion mutants of the
-adrenergic and
muscarinic M1 receptors were internalized to a similar extent compared
with the WT receptors (4, 30).
The hPAFR contains two putative internalization motifs in its sequence
289DPVIYCFL296
at the terminus of the 7th transmembrane domain. The sequence (N/D)P(X)2,3Y was proposed as an internalization
motif by analogy with the endocytic motif NPXY (52) found in
low density lipoprotein, transferrin, epidermal growth factor, and
insulin receptors. However, the role played by this motif is not as
well defined in G-protein-coupled receptors since substitution of some
of these residues may affect other functions such as G-protein coupling
(37, 38). We have shown that this is the case for the hPAFR with the
substitutions of Asp289 (41) and Tyr293 to
alanine. On the other hand, a substitution by their isosters Asn and
Phe, respectively, barely affected the coupling and only slightly
influenced the kinetics of internalization. Therefore it is the
structure of the amino acid that is important, not its polarity or side
chain charge, indicating that these residues may form hydrogen bonds
with others in the vicinity. As already suggested (38), the residues of
this motif seem to participate in the preservation of an adequate
conformation of the receptor and do not directly participate in
internalization. This conclusion is supported by the fact that the
mutant Y293A allows for the endocytosis of 3H-PAF just as
efficiently as the WT receptor. The lack of effect of the Y293A
substitution on internalization also indicates that the other putative
internalization motif YXX does not have such a function
in the hPAFR.
The molecular mechanisms responsible for receptor-mediated endocytosis are still poorly understood. The potential involvement of PLC activation in the internalization process was suggested following the discovery that the complex AP-2 could bind to phosphatidylinositol (4,5)bisphosphate (53) and inositol (1,4,5)trisphosphate (54). It has also been shown by pertussis toxin treatment that coupling and activation of the G-protein was important for the internalization of somatostatin receptors (55). Despite the fact that loss of functional coupling seems to correlate with a decreased internalization for other receptors (3, 31) and for most of our uncoupled mutant receptors (D63N, A230E, and D289A), results obtained with pertussis toxin treatment and the Y293A mutant indicate that G-protein coupling and signalization do not participate in hPAFR internalization. Similar discrepancies between internalization and signalization were also observed for muscarinic receptors (56) and for the type 1 angiotensin II receptor (57).
Moreover, results with an activator and inhibitors of PKC show that
phosphorylation by this kinase does not trigger or modulate the
internalization process. However, for some receptors, phosphorylation by a second messenger-dependent kinase might play a role in
this process. It has been reported for the neurotensin receptor that the inhibition of PLC by U-73122 impaired the agonist-induced internalization of this receptor in transfected CHO cells and in
N1E-115 neuroblastoma cells (58). Phosphorylation could also increase
the probability of a protein to become a substrate for GRK2 (59), and
in certain circumstances, the action of these kinases could modulate
the internalization process (41, 60, 61). In addition, some GRKs can be
influenced by phosphorylation cascades, as GRK2 activity and
translocation can be increased through direct activation of PKC (62).
GRK1, -2, and -3 are cytoplasmic kinases that translocate to the plasma
membrane after a stimulus-mediated receptor activation. The
translocation and activation of GRK2 and -3 is dependent on their
association with specific G-protein subunits (63, 64) via their
pleckstrin homology domains (65), in contrast to GRK5 and -6 that are
associated with the membrane. The actions of these kinases are
therefore dependent on G-protein coupling, and this could explain why
the internalization of some receptors (55), but not of others, like hPAFR and type 1 angiotensin II receptor (57), is enhanced by G-protein
activation as this might depend on which GRK will phosphorylate the
activated receptor in a given cell type.
Our study indicates that ligand-induced signalization and the motifs
(N/D)P(X)2,3Y and YXX are not
involved in the process of internalization, whereas the use of
internalization inhibitors suggests the participation of
clathrin-coated vesicles in hPAFR internalization. Recently, it has
been shown that arrestins could play a role in internalization by
acting as adapter proteins between the receptors and clathrin and that
GRK-mediated phosphorylation could augment this interaction (59-61).
The possibility that this mechanism is involved in the internalization
of hPAFR is presently under investigation in our laboratory.
We thank D. Gingras and S. Turcotte for excellent technical assistance.