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-Adaptin Association
INSERM U344 (L.V., A.P., M.E.) Faculté de Médecine
Necker F-75730 Paris, Cedex 15, France
Leuven Poultry
Research Group Zoological Institute (L.V., E.R.K.) and
Department of Animal Sciences (E.D.) Katholieke Universiteit
Leuven B-3000 Leuven, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-adaptin, a subunit of
adaptor protein AP-2, associates with the GHR upon hormone stimulation.
The clathrin-coated pit pathway therefore seems to be involved in the
endocytosis of cGHR, as AP-2 is known to intervene in the recruitment
of receptors to these pits. Interaction with -adaptin may occur
through a common epitope of the chicken and mammalian GHR, since
receptors from both species bind similar amounts of -adaptin;
alternatively, two different epitopes with similar affinity may be
involved. Therefore, not -adaptin but an uncharacterized factor,
presumably interacting with the identified ß-turn endocytic code, is
responsible for the difference in internalization kinetics. Finally,
the present study illustrates that functional amino acid motifs of
receptors can be derived from comparative studies. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A characteristic of receptors for polypeptide hormones is their ability for receptor-mediated endocytosis (5). Internalization starts with the recruitment of hormone-receptor complexes into clathrin-coated pits by AP-2, a plasma membrane-specific, heterotetrameric adaptor protein (6). Several cytoplasmic motifs, called endocytic codes or internalization signals, are required for efficient internalization and/or AP-2 association (7, 8). Studies with rat GHR (rGHR) deletion mutants have indicated that the first 90 amino acid (aa) residues of the cytoplasmic domain contain endocytic codes, of which Phe346 has been identified (9). A Leu-pair and a tetrapeptide predicted to adopt a ß-turn were suggested to be internalization signals for the short isoform of the rat PRL receptor (srPRLR) (10). Ligand-mediated endocytosis, however, appears not to be a prerequisite for GH-induced gene transcription mediated by the JAK/STAT signal transduction pathway (9).
Our interest in the internalization of GH and GHR arose from studies in which hepatic GHR capacity was reduced after injecting GH into hypophysectomized chickens (11). Fractionation experiments of adult hen liver indicated that a major portion of GHR was allocated to an intracellular compartment (12). A detailed in vitro study of internalization kinetics, the first in a nonmammalian species, became possible by the cloning of the chicken GHR (cGHR) (13). Moreover, analysis of the GHR amino acid sequence suggested that an endocytic code identified in the srPRLR (10) was present in mammalian GH receptors but not in the cGHR, making the cGHR an interesting model for the study of receptor-mediated endocytosis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. By Scatchard analysis, properties of
binding were calculated. Since the heterologous human GH (hGH) is a
better competitor of [125I]cGH than cGH,
[125I]hGH was used for further experiments. Dissociation
constant (Kd) for hGH is 0.50 ± 0.06 nM
(n = 4), which is within the range reported for binding to the GHR, in
a similar transfection study or in hepatic microsomal fractions
(14, 15, 16). On the average, 1,000,000 binding sites for hGH are found per
transfected cell.
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). At t = 0 min, cell
surface binding values for rGHR and cGHR are identical, 17% and 20%,
respectively. Internalization by the cGHR is slower as compared with
the rGHR. After 15 min at 37 C, label internalized through the cGHR is
barely detectable, whereas internalization in the rGHR-transfected
cells has already started (P < 0.01). After 1 h
at 37 C, internalization by the cGHR is 40% lower than by the rGHR
(P < 0.0001). When cGHR-transfected cells are left
longer at 37 C, the curve flattens and does not attain rat maximal
levels (results not shown). Labeled hGH and cGH are internalized
equally by the cGHR (results not shown).
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). In the rGHR, seven tetrapeptides are
predicted to adopt a ß-turn configuration by the Chou-Fasman
algorithm (17). In the rGHR, as well as in the cGHR, the first ß-turn
is predicted with the highest probability and ends in two vicinal Leu
(Asp311-Pro312-Asp313-Leu314-Leu315).
The next three ß-turns are positioned between aa 330 and 344 and are
part of one region contiguous with Phe346. However, the
middle ß-turn of these three is not present in chicken due to a
triple mutation. In this ß-turn, a Pro is in second place in the
rGHR, which, of all amino acids, has the strongest ß-turn potential
on this position. Consequently, replacing Pro335 by
Thr307 reduces ß-turn potential of the cGHR. As the third
residue Asp336 is changed to Gln308, a ß-turn
becomes even more improbable. The fourth residue does not affect
ß-turn potential.
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), in
agreement with previous results (20). Signal transduction and
activation of transcription are similar for the wild-type and
Flag-tagged GHR as well. GH-dependent induction of luciferase activity
is used to assay bioactivity. Furthermore, indirect immunofluorescence
studies using Flag-cGHR-transfected cells showed that the
epitope-tagged receptor is internalized to cell compartments that
participate in the endocytic pathway (data not shown). Indeed, previous
studies with N- and C-terminally Flag-tagged PRLR have shown that
epitope tagging does not significantly change bioactivity, binding
properties, or internalization behavior (24).
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).
Phosphorylation on tyrosine residues of JAK2 and STAT5 was analyzed for
the rat and chicken GHR (Fig. 4). JAK2- and
STAT5-containing complexes were precipitated from cell lysates using
specific anti-JAK2 or anti-STAT5 antibodies. Tyrosine phosphorylation
was then evaluated by SDS-PAGE in reducing conditions, followed by
probing of the obtained blots with a specific monoclonal
antiphosphotyrosine antibody. Tyrosyl phosphorylation of JAK2
(Mr 130,000) is induced by GH, as early as 15 min after GH
addition. Later on, GH-dependent tyrosyl phosphorylation of JAK2
decreases. Similarly, activation of STAT5 (Mr 95,0000) by
GH is maximal at 15 min. Although internalization of the cGHR is
reduced, this does not inhibit the initial steps of signal transduction
by the JAK/STAT pathway, i.e. activation of tyrosine kinase
JAK2 and of STAT5 during the same time course. On the contrary, tyrosyl
phosphorylation of STAT5 appears prolonged for the cGHR, as compared
with rabbit GHR (rbGHR).
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-Adaptin in GHR Internalization-adaptin, a component of adaptor protein AP-2 (Fig. 5). COS-7 cells expressing either Flag-cGHR
or a mammalian GHR [Flag-tagged rbGHR (Flag-rbGHR)] were analyzed for
their association with -adaptin upon hormone stimulation. Immunoblot
analysis of Flag-GHR complexes that were purified using
immunoprecipitation with an anti-Flag antibody reveals that a specific
band of apparent Mr of 100,000 corresponds to -adaptin
and that it displays a GH-dependent association with the GHR. The
amount of -adaptin associated with the cGHR is at least as large as
that associated with the rbGHR.
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Pro307-Asp308). Mutation of the potential
endocytic code did not affect binding characteristics of hGH
(Kd, 0.4 nM; 21% specific binding; on the
average 910,000 binding sites for hGH are found per transfected cell).
Notwithstanding the different endocytic behavior of the rat and
wild-type chicken GHR, internalization rates of the mutant cGHR and the
rGHR were similar, confirming the relevance of the mutated ß-turn for
receptor-mediated endocytosis of GH (Fig. 6).
Activation of gene transcription by the JAK/STAT pathway was identical
(luciferase activity induction factor of 9.1 ± 0.8 for wild-type
cGHR vs. 9.5 ± 0.5 for
Pro307-Asp308 cGHR mutant). The association of
the mutant to -adaptin was similar to that of wild-type cGHR (data
not shown). Furthermore, the pivotal role of Pro335 for
optimal internalization of the rGHR was corroborated by alanine
scanning of the
Lys334-Pro335-Asp336-Phe337
tetrapeptide in the rGHR. The Pro335 Ala335
rGHR mutant internalized less (t = 60 min, P <
0.01) than Lys334 Ala334,
Asp336 Ala336, and Phe337
Ala337 mutants that are very similar in internalization
compared with the wild-type rGHR (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-adaptin, a subunit of the AP-2 complex that initiates
formation of clathrin-coated pits (6). The identified endocytic code
was not involved in ligand binding, -adaptin association, or
activation of JAK/STAT. Although internalization of the cGHR, as compared with mammalian GH receptors, was reduced, gene transcription through the JAK/STAT signal transduction pathway was activated by the cGHR. Transcriptional activity mediated by STAT5 was evaluated with a reporter gene. The gene construct contains lactogenic hormone response element (LHRE) coupled to the thymidine kinase minimal promoter and the luciferase gene. LHRE is the element of the ß-casein promoter that was used for affinity purification of STAT5 (25). Moreover, the level and timing of tyrosine phosphorylation of JAK2 of the cGHR and rGHR did not differ. Tyrosyl phosphorylation of STAT5 seemed even prolonged. If the cGHR has a longer half-life on the plasma membrane, then STAT5 has more time to bind to phosphorylated tyrosines within the GHR and will be more phosphorylated by JAK2. Distinction of internalization capacity and signal transduction was previously evaluated at the molecular level; different cytoplasmic regions of the GHR sequence are responsible for these processes. Proximal to the membrane, the GHR contains a Pro-rich region, called Box 1 (aa 297–311 in rGHR), that is conserved in the cytokine/GH/PRL receptor family and is required for signal transduction by the JAK/STAT pathway (16). On the other hand, the cytoplasmic region of the rGHR ranging from amino acid residue 318–380 is involved in internalization. More specifically, Phe346 was identified as being critical for GH-dependent internalization of the rGHR, but not for activation of gene transcription by JAK/STAT (9).
Sequences of internalization signals were first elucidated for the low-density lipoprotein receptor, the transferrin receptor, and the cation-independent mannose-6-phosphate receptor. Although endocytic codes of these receptors differ in specific sequence, they all share a common three-dimensional conformation and chemistry and form tight (or ß-) turns. For the transferrin receptor, it was reported that a Tyr-containing ß-turn internalization signal could be replaced by Leu-Leu, which suggests that these two signals are functionally equivalent (7). Recently it was reported, however, that the di-Leu motif is solely involved in the internalization of truncated GHR or PRLR (srPRLR), but not of the full-length GHR (26).
Secondary structure analysis of the juxtamembranous cytoplasmic region of the GHR with high interspecies homology revealed the presence of several peptide motifs, predicted to adopt a ß-turn configuration, the first tight turn having the highest probability found in our analyzed region. In the region important for efficient internalization by the rGHR (aa 318–380), three contiguous ß-turn tetrapeptides occur within a highly conserved stretch of 15 aa. The middle ß-turn of this trio, however, is not present in chicken, as Pro307 and Asp308 are replaced in cGHR by residues with less ß-turn potential. The fourth residue, Phe309, is not conserved either, but this mutation does not affect ß-turn probability. Moreover, by Ala mutation, Phe309 was previously shown not to be required for internalization (9). Finally, the rGHR Phe346 identified previously as an internalization motif (9) is conserved in the cGHR. The reduced internalization capacity of the cGHR may therefore be due to the different three-dimensional structure of the receptor molecule, which hinders endocytosis. The current vision is that these ß-turn internalization motifs are exposed on the surface of the cytoplasmic tail, enabling interaction with adaptor molecules (7). Deletion of a similar motif (Leu-Pro-Gly-Gly) in the srPRLR caused a 50% reduction in internalization (10), which corresponds to the diminished internalization of cGHR, as compared with rGHR. The remaining internalization capacity of the cGHR could then be due to endocytic codes common to both species.
Little is known about the underlying mechanisms controlling GHR
internalization and trafficking. In general, receptor-mediated
endocytosis involves the concentration of receptors in clathrin-coated
pits with the help of a plasma membrane-associated adaptor protein
AP-2. This protein complex consists of four subunits: 2- and
ß-adaptin of 100 kDa, a µ2-chain of 50 kDa, and a small 
2-chain
of 16 kDa (6). Upon ligand stimulation, -adaptin associated with the
srPRLR (10). Our results demonstrate, for the first time, the
GH-inducible binding of a GHR to -adaptin.
Furthermore, we compared the interaction of -adaptin with chicken or
mammalian GHRs, in view of the species-dependent internalization
behavior. As both receptors bind similar amounts of -adaptin, our
experiments indicate that cytoplasmic motifs required for
internalization and for -adaptin binding are not necessarily the
same. Further experiments are necessary to determine which region(s) of
the GHR is (are) responsible for -adaptin association. In
vitro studies showed that Tyr-containing sorting signals interact
with the µ2-subunit of AP-2 (27). The existence of distinct motifs
for internalization and AP-2 association, respectively, was reported
for the receptor of epidermal growth factor. Kinetics of
internalization of a mutant receptor for epidermal growth factor
lacking the Tyr-containing AP-2 binding site were indistinguishable
from those of its wild-type counterpart and were independent of AP-2
(28).
Although there is no doubt that AP-2 is involved in clathrin-dependent
endocytosis, the previous and present studies suggest that -adaptin
may be necessary, but not sufficient, for a maximal response. The
ß-turn lost in cGHR may interact with ß-arrestin that induced a
concentration of the ß2-adrenergic receptor in
clathrin-coated pits (29). Furthermore, mammalian and chicken GHR may
differ in ubiquitin association. Recently it was shown that GHR
ubiquitination is not only a prerequisite for GHR degradation, but also
for ligand-dependent endocytosis (30). Moreover, the ubiquitin
conjugation and ligand-induced internalization are coupled events,
since they are both disrupted by the F345A mutation (31). Finally, GHBP
may interfere with GHR internalization. The culture medium of cells
transfected with the cGHR contains large amounts of GHBP, since cGHBP
is generated by proteolysis of the full-length GHR (32), contrary to
rGHBP that is not detectable in medium of rGHR-transfected cells (33).
The formation of GH.GHR.GHBP heterotrimers may hinder internalization
of the cGHR.
In conclusion, our study shows that 1) the cGHR is internalized, a
process presumably mediated by the clathrin-coated pits endocytic
pathway, 2) reduced internalization of the cGHR as compared with
mammalian GH receptors is related to the loss of a conserved
cytoplasmic internalization motif predicted to adopt a ß-turn, and 3)
the level of internalization is not correlated to the level of
-adaptin association or activation of gene transcription.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Dr. J. Burnside (University of Delaware, Newark, DE) kindly provided the cDNA encoding the cGHR cloned in the pSG5 expression vector that is under SV40 transcriptional control (36). The rGHR expression plasmid pLM108, a pUC8 construct containing the human metallothionein IIa promoter and SV40 enhancer, was donated by Dr. G. Norstedt (Center for Biotechnology, Karolinska Institute, NOVUM, Huddinge, Sweden) (37).
Construction of Flag-Tagged cGHR
To enhance immunoprecipitation and immunodetection, the cGHR was
epitope-tagged at the N terminus with an octapeptide, named Flag
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Monoclonal antibodies directed
against the Flag-epitope are commercially available (Eastman Kodak Co., Rochester, NY). The cDNA encoding the mature
wild-type cGHR was inserted in the pFlag-CMV-1 expression vector
(Eastman Kodak Co.), after the preprotrypsin signal
peptide sequence that ensures correct membrane-bound expression. By
oligonucleotide-directed mutagenesis using PCR, a cGHR insert was
produced, excluding the original signal peptide (the first 16 aa) and
introducing at the 5'- and 3'-end unique restriction enzyme sites
recognized by NotI and XbaI. An N-terminally
Flag-tagged cGHR construct (pFlag-cGHR) was obtained after enzyme
digestion, ligation, and ampicillin selection in Escheria
coli DH5 cells. The presence of the Flag epitope in the engineered
cGHR was confirmed by dideoxynucleotide sequence analysis (38). The
rbGHR inserted in pFlag-CMV-1 vector (pFlag-rbGHR) was obtained from S.
Moutoussamy (INSERM U344) (22).
Construction of Flag-Tagged cGHR (T307P,Q308D) and rGHR Mutants
(K334A; P335A; D336A; F337A)
Substitutions of Thr307 and Gln308 by
Pro and Asp, respectively, were obtained using the Flag-CMV-1-cGHR
plasmid. A single-stranded DNA was generated by using the origin of
replication of the M13 phage present in the vector, in the
Escheria coli CJ236 strain in the presence of M13K07 helper
phage. This single-stranded DNA was then used as a template for
oligonucleotide-directed mutagenesis with the primer
5'-CATTGTATAGGTCTGGCTTGTAGTTG-3'. A similar strategy was used to
construct K334A, P335A, D336A, and F337A mutants. The modified regions
were verified by sequencing (38).
Cell Culture and Transfection
COS-7 cells were grown as monolayers in DMEM containing 10%
FCS, 2 mM L-glutamine, 50 IU/ml penicillin, and
50 µg/ml streptomycin. Routinely, cells were cultured at 37 C, in a
humid 5% CO2 incubator. At 70–80% confluence, cells were
transfected by the diethylaminoethyl-dextran/chloroquine method. Four
hours after addition of DNA precipitates, cells were subjected to
Me2SO shock, washed, and cultured in fresh complete medium
for 48 h (10).
Analysis of GHR Binding
Hormone binding to transfected COS-7 cells was performed in
six-well plates. Before binding, cells were washed with DMEM and kept
in serum-free medium for at least 4 h. Subsequently, plates were
put on ice and washed with ice-cold HEPES binding buffer (HBB: 25
mM HEPES, 124 mM NaCl, 4 mM KCl, 1
mM CaCl2, 1.5 mM MgCl2,
and 2 mM KH2PO4, pH 7.4). In a
final volume of 1 ml HBB containing 1% BSA (Fraction V, Sigma Chemical Co., St. Louis, MO) (HBB-BSA) whole cells were
incubated with 80,000–100,000 cpm [125I]GH. At the end
of incubation, unbound label was removed by two HBB washes; 1 ml 1
N NaOH was added, and radioactivity in lysates was measured
using a 
-counter. Specific binding was determined by subtracting the
amount of [125I]GH bound in the presence of excess
unlabeled GH (91 nM). For Scatchard analysis, increasing
amounts of unlabeled GH were added to compete with
[125I]GH for binding in saturating conditions (overnight
at 4 C). With LIGAND software (Elsevier-BioSOFT, Cambridge, UK),
Kd and binding capacity were calculated (39). Data are
represented as means ± SEM.
Internalization Studies
Internalization was analyzed as described by Allevato et
al. (9). Briefly, cells transfected with 0.1–1 µg cDNA were
treated as described for binding analysis. Cells were incubated with
70,000–100,000 cpm [125I]GH at 4 C to bind cell surface
receptors (hGH for 2.5 h, cGH for 6 h); the low temperature
prevented label internalization. Unbound ligand was removed by washing
twice with ice-cold HBB; culture plates containing 1 ml HBB-BSA were
then transferred to 37 C for various times; surface-bound
[125I]GH was removed by a 3-min exposure to an acid wash
buffer (150 mM NaCl and 50 mM glycine, pH 2.5),
and cells were lysed to recover acid-resistant binding. For each time
point, internalization was expressed as the percentage of specific
intracellular radioactivity toward specific binding at t = 0 min.
Data were statistically analyzed with SAS software (SAS Institute, Inc., Cary, NC) using the general linear model
procedure and are represented as means ± SEM.
Secondary Structure Prediction
The occurrence of ß-turns was predicted using the Chou-Fasman
algorithm (17). ß-Turns are chain-reversal regions consisting of
tetrapeptides. Based on x-ray crystallography data of 29 proteins, the
average overall frequencies to be part of an -helix
(<f> = 0.38), a ß-sheet (<fß> =
0.20), or a ß-turn (<ft> = 0.32) were determined.
Conformational parameters Pt, P, and
Pß were obtained for all 20 amino acids, by expressing
their frequency (f, fß, and
ft) relative to the respective average frequency. Strong
ß-turn formers are Asn, Gly, and Pro, all with Pt >
1.50. In addition, the bend frequency (f) of each amino acid was
calculated for the four positions of the ß-turn. The probability of a
bend starting at residue i is then p(t) = fi x
fi+1 x fi+2 x fi+3. To
predict whether a tetrapeptide is a ß-turn or rather part of an
-helix coil or ß-sheet, Pt, P, and
Pß are averaged over the four residues. A ß-turn is
predicted if p(t) > 0.75 x 10-4,
{Pt} > 1 and {P} <
{Pt} > {Pß}.
Luciferase Bioassay
To test biological activity of Flag-tagged and wild-type
receptors, a 293-cell bioassay was used (40). A 6-well plate was
transfected by the calcium phosphate technique using, per well, 33 ng
of receptor plasmid, 17 ng of a ß-galactosidase expression vector
(pCH110, Pharmacia Biotech, Uppsala, Sweden), and 250 ng
of LHRE-tk-Luc. The latter is a construct that contains a GH-responsive
promoter [thymidine kinase (tk) minimal promoter and 6 repeats of the
LHRE of the ß-casein promoter] fused to the firefly luciferase
reporter gene. One day after transfection, cells were incubated
overnight with 0 or 23 nM hGH serum-free medium. Cell
extracts were prepared and enzyme activities were determined. Activity
of ß-galactosidase was used to normalize luciferase expression levels
for differences in transfection efficiency.
Immunoprecipition and Western Blot Analysis
COS-7 cells were grown on 100-mm dishes, transfected with
Flag-tagged receptor plasmid (1 µg/dish), cultured overnight in
serum-free medium, and stimulated (or not) with 45 nM hGH
(20 min at 37 C). Cellular proteins were extracted in 1 ml of lysis
buffer (50 mM Tris, 2 mM CaCl2, 100
mM NaCl, 8% glycerol, 0.8% Triton X-100, pH 7.6)
containing phosphatase and protease inhibitors (1 mM
o-Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 2 µg/ml
leupeptin, 1 µg/ml pepstatin). Lysates were incubated overnight at 4
C with agarose beads bearing Flag monoclonal antibodies (M1) for
precipitation of receptor complexes. Immunoprecipitated complexes were
washed with fresh cold lysis buffer, boiled in SDS sample buffer, and
subjected to 7.5% SDS-PAGE. Proteins were then transferred onto a
polyvinylidene difluoride membrane (PVDF, Polyscreen, Dupont NEN, Boston, MA), and blots were incubated for 2 h at room
temperature with monoclonal antibodies directed against either Flag
(M2, 0.5 µg IgG/ml) or -adaptin (AC1-M11, 1:100, Affinity BioReagents, Inc. Neshanic Station, NJ). Finally, membranes
were incubated for 1 h with alkaline phosphatase-linked goat
antimouse secondary antibody (1:10,000), and proteins were revealed
using the Vistra ECF Western blotting system (Amersham Pharmacia Biotech, Little Chalfont, UK).
To evaluate JAK2 activation by GH, 293 cells were cotransfected with GHR (4 µg) and human JAK2 (2 µg) cDNA. For the determination of GH-dependent induction of STAT5 tyrosyl-phosphorylation, cells were transfected with GHR (4 µg/ml, JAK2 (0.1 µg/ml), and STAT5 (2 µg/ml) cDNA. JAK2 and STAT5 expression vectors were kindly provided by Drs. J. Ihle and B. Groner, respectively. Cell lysates were incubated with the designated antibody [anti-JAK2 (1 µg/ml; Upstate Biotechnology, Inc., Lake Placid, NY) or anti-STAT5 (1 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)] and protein A-Sepharose (50%, vol/vol). Finally, immunoprecipitated complexes were analyzed by Western blot, using a monoclonal antiphosphotyrosine (4G10; Upstate Biotechnology, Inc.; 1:4,000), a polyclonal anti-JAK2 antibody (1:5,000), or a monoclonal anti-STAT5 (1:1,000) antibody. Finally, the membranes were incubated with an antirabbit or antimouse IgG-conjugated horseradish peroxidase (1:8,000) and revealed by the enhanced chemiluminesence (ECL) detection system (Amersham Pharmacia Biotech).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Fund for Scientific Research-Flanders (Belgium) (F.W.O. G.0235.97).
Received for publication October 19, 1998. Revision received July 3, 1999. Accepted for publication July 26, 1999.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) concentrations. b, The data in panel a were used to
perform Scatchard analysis.
