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Original Studies |
INSERM U-344, Endocrinologie Moléculaire, Faculté Necker-Enfants Malades (J.W., N.E., M.-C.P.-V., J.F.), 75015 Paris, France; Howard Hughes Medical Institute and Department of Genetics, Stanford University Medical Center (M.A.B., U.F.), Stanford, California 94305; the Department of Pediatrics, University of California Children’s Hospital (M.E.G.), Los Angeles, California 90095-1752; the Department of Pediatrics, King Faisal Specialist Hospital (N.S.), Riyadh 11211, Saudi Arabia; the Department of Pediatrics, Baystate Medical Center Childrens Hospital (E.O.R.), Springfield, Massachusetts 01199; and the Division of Molecular and Genetic Medicine, The Medical School, University of Sheffield, Royal Hallamshire Hospital (S.D.), Sheffield, United Kingdom S10 2JF
Address all correspondence and requests for reprints to: Dr. Joëlle Finidori, INSERM U-344, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. E-mail: finidori{at}necker.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The GHR, which belongs to the large family of cytokine receptors (6, 7) exists in two forms: the membrane-bound receptor and the soluble GH-binding protein (GHBP) that corresponds to the extracellular domain of the membrane receptor (8, 9). Binding of GH to its receptor induces receptor dimerization (10, 11, 12) and activation of the tyrosine kinase JAK2 (13), which results in activation of members of the signal transducer and activator of transcription (STAT) family (14). Stat5b could be the major STAT protein activated by GH in liver (15).
The GHR gene spans more than 87 kbp of the short arm of human chromosome 5 and includes 9 coding exons and several alternatively spliced noncoding exons in the 5'-untranslated region (3). Exon 2 encodes an 18-amino acid signal sequence that is cleaved from the mature protein. Exons 3–7 encode the ligand-binding extracellular domain of the receptor. Exon 3 deletion occurs by alternative splicing, which is genetically transmitted (16) and does not modify normal GH-binding activity. In addition to GH binding, residues in the extracellular domain participate in dimerization in response to binding to a single GH molecule. Exon 8 encodes the transmembrane domain. Exons 9 and 10 encode the cytoplasmic, signal-transducing domain, including a proline-rich region (box1).
A large number of different GHR gene defects have now been reported in individuals affected with LS, including nonsense mutations, splice site mutations, a multiexon deletion, dinucleotide deletions, and missense mutations. Most were reported in reviews (17, 18, 19). Other novel mutations were recently published (20, 21, 22, 23, 24, 25). In most cases, the mutations are hypothesized to be functional null alleles, and the GHR gene defect results in lack of binding activity of the mutant GHR/GHBP. However, 25% of the patients with LS have normal or elevated serum GHBP (19); in these cases, receptor dimerization or intracellular signaling events could be impaired, resulting in the GH resistance. Two cases of GHR insensitivity caused by heterozygous splice site mutations, resulting in a truncated receptor missing most of the intracellular domain, appear to act as dominant negatives (26, 27).
We have analyzed the GHR genes of additional unrelated individuals affected with LS and have identified missense mutations of each of four adjacent amino acids in the extracellular domain and a truncating mutation in the intracellular domain. One of the four adjacent substitutions (D152H) has been described (28, 29); the three others are novel (I153T, Q154P, and V155G).
In this report we study in 293 transfected cells the consequences of the three new mutations reproduced in the human GHR complementary DNA (cDNA). We show that the cell surface expression of these three mutants is impaired and is associated with variable defects in GH binding affinity. These results are compared with the consequences of the previously studied D152H mutation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patient 1. Patient 1 was the first case of LS in whom GH insensitivity was demonstrated (30). Growth retardation had been noted during the first postnatal year. At age 30 yr, he was well proportioned, with a height of 118 cm and weight of 31 kg. Basal plasma GH levels were 5–10 ng/mL on four separate occasions (normal range, 1.8 ± 0.41 ng/mL). His GH level was not suppressed by oral glucose administration and was further augmented by insulin-induced hypoglycemia and arginine infusion. His response to exogenous human GH was attenuated with respect to serum urea nitrogen; urinary nitrogen, calcium, and phosphorus excretion; and insulin responsiveness. When last seen, he was 42 yr old, sexually mature, and had fathered two children. As the patient was lost to follow-up, no further information is available. GHR mutation detection studies were performed on frozen cells (31).
Patient 2. This boy was first evaluated at age 30 months because of small size, stubby appearance, and a history of hypoglycemia. He had high random GH levels during sleep (68 ng/mL). After stimulation with L-DOPA and arginine, his GH levels were 19.8 and 17.5 ng/mL, respectively. As his growth failure continued, he was treated with exogenous GH under the hypothesis that his own GH may be biologically inactive. GH therapy, however, did not result in an increase in growth rate. At age 6 yr, his overnight pooled GH level was 16.9 ng/mL (Hybritech, La Jolla, CA), insulin-like growth factor I (IGF-I) was 0.1 U/mL, and GHBP determined by an undefined method was said to be "36% of the adult normal standard, a level which was considered normal for a child his age." He subsequently responded to treatment with IGF-I by significantly increasing his growth rate.
Patient 3. At age 23 months, this patient’s height age was 7 months, and a somatomedin C level was slightly low at 0.48 U/mL. At age 2 yr 8 months, unstimulated and clonidine-stimulated GH levels were elevated (42.8 and 93.8 ng/mL, respectively), and a somatomedin C level was below 0.1 U/mL. A 6-month trial of GH therapy at age 6 yr was associated with a poor growth response. By a GH binding assay, her plasma GHBP levels were 1.2% and 6.1% (normal age-matched controls: mean, 87%; range, 34–154%). In 1993, GHBP was 143 pmol/L (normal range, 431-1892 pmol/L), as determined by a commercial laboratory (Endocrine Sciences, Inc., Tarzana, CA). IGF-I therapy from ages 10–13 yr has been associated with improved growth velocities of 9.8 cm/yr in the first year and 5.8–6.0 cm/yr in the subsequent 2 yr. After about 18 months of IGF-I therapy, the patient developed a slipped capital femoral epiphysis, which has been treated surgically. Maternal height was 159.4 cm, and paternal height was 164 cm.
Patient 4. Patient 4’s birth height was 46 cm, and at age 1 yr, her height age was 4 months (60 cm). When evaluated at 20 months, she had a prominent forehead, blue sclerae, malar hypoplasia, and very small hands and feet. Her bone age was delayed. She had borderline hypoglycemia (41 mg/dL) during fasting. Her baseline GH level of 41 ng/mL rose to 130 ng/mL upon clonidine stimulation. Her somatomedin C level was in the normal range, and an IGF-binding protein-3 level was low (732 ng/mL; measured by Kabi, Stockholm, Sweden). GHBP levels were not determined. Paternal height was 172.6 cm, and maternal height was 162.1 cm.
Screening for GHR gene mutations
Genomic DNA was extracted from fresh blood samples or transformed leukocytes by standard procedures. In some cases, only blood spot samples on filter paper were available, which were analyzed directly without DNA extraction. PCR amplification and denaturing gradient gel electrophoresis (DGGE) of the nine coding exons and intron-exon boundaries of the GHR gene were performed using procedures previously described (17, 32), except for redesign of some amplification primers. The new reverse primer for exon 2 amplification is 5'-GAA TAC AGT TCA GTG TTG TTT-3'. For exon 3, the forward primer (5'-GAT GGA CTA GAT GGT TTT GCC TTC CTC TTT CTG TTT CAG-3') and reverse primer (GC-clamp-5'-GGA TAG TAG CTT AAT TAC ACT AAA ACA TGG-3') are both newly designed.
Because heteroduplex bands are more easily detected by DGGE, we first analyzed available parents who are obligate heterozygous carriers of a GHR mutation resulting in LS. If parental samples were unavailable, 25 µL exon-specific PCR products from a proband and a normal control were mixed, denatured, and reannealed to allow the formation of heteroduplexes before electrophoresis.
DNA sequencing
Double stranded PCR products from exons showing abnormal bands on DGGE and from intron 9 were sequenced as described previously, using the Circumvent Thermal Cycle Sequencing Kit (New England Biolabs, Inc., Beverley, MA). Haplotypes were assigned based on the sequence variants in intron 9, according to a previously described nomenclature (33).
Restriction enzyme analysis
Exon-specific PCR products from a panel of 55 normal controls and available relatives of patients were tested for the presence of the identified mutations that altered restriction enzyme sites in exon 6 (EcoRV, BstXI, and StyI). Digested PCR products were electrophoresed in 4% NuSieve (Rockland, ME) agarose gels and visualized by ethidium bromide staining.
Construction of expression plasmids
Site-directed mutagenesis experiments were performed from the
full-length human (h) GHR cDNA subcloned in the pcDNA1 vector (34) to
produce mutant GH receptors. Oligonucleotides 5'-G ATT CAT GCA GAT
ACC CAA GTG AGA TGG GAA G-3', 5'-G ATT CAT GCA GAC
ATCCCA GTG AGA TGG GAA G-3', and 5'-G ATT CAT GCA GAC ATC
CAA GGG AGA TGG GAA G-3', corresponding to
nucleotides 540–571, were used to build mutant hGH receptors (Mutated
nucleotides are underlined.) This results in the substitution of
isoleucine to threonine at position 153 (ATC
ACC), glutamine to
proline at position 154 (CAACCA), and valine to glycine at position
155 (GTGGGG). These mutations were confirmed by DNA sequencing.
Cell culture, transfection, and binding assays
Human 293 cells were grown and transfected as previously
described, using the calcium phosphate method (35). Twenty-four hours
after transfection, the cells were serum-starved for 12 h. For
cell surface binding assays, cells (3 x 106
cells/six-well plate) were washed twice with PBS containing 1% BSA and
incubated with [125I]hGH (105 cpm/well)
overnight at 4 C in the absence or presence of unlabeled hGH in various
concentrations. The cells were then washed with the same buffer and
solubilized in NaOH (1 N) for counting. Total particulate
membrane fractions were prepared as follows. The cells (6 x
106 cells/P100 petri dish, Falion, Plymouth,
England) were harvested in PBS with 1% BSA and homogenized by Polytron
(Kinematica, Luzerne, Switzerland). Membrane fractions were
separated from cytosol by ultracentifugation at 100,000 x
g (30 min at 4 C). The pellet was resuspended in 500 µL of
25 mmol/L Tris-20 mmol/L MgCl2, pH 7.4, solution,
and the amount of protein was quantified by Lowry assay. Membrane
fractions (200 µg protein) were incubated overnight at 4 C in 300
µL of the same buffer containing 0.1% BSA, 105 cpm
[125I]hGH, and various amounts of unlabeled hGH. The
reaction was then stopped by adding 300 µL buffer (at 4 C, with 0.1%
BSA). Bound and free hGH were separated by two centrifugations at
17,000 x g (the pellet was washed with the same buffer
after the first centrifugation) and specifically bound
[125I]hGH in the pellet was determined using a
-counter. Scatchard analyses were performed with the program Ligand
(36).
Immunoprecipitations and Western blotting
hGH was biotinylated using the Boehringer kit (Boehringer Mannheim, Indianapolis, IN) at a molar ratio of 1:5 (recombinant hGH was obtained from Ares-Serono Laboratories, Inc., Geneva, Switzerland). Affinity purification of complexes formed between biotinylated hormone and the wild-type (WT) or mutant receptors that were either present at the cell surface or in total cellular compartments was performed on parallel sets of 293 cells (106 cells/P100 petri dish) transfected with 5 µg cDNA/P100 dish; 24 h after transfection, the cells were serum-starved for 12 h, and the cell surface labeling was performed by incubating one set of cells with biotinylated hGH (2 µg/mL in PBS-1% BSA) for 2 h at 4 C before washing with PBS and then lysis with Triton. Total receptor labeling was performed on the other set of transfected cells, which was first lysed and incubated with biotinylated hGH (2 µg/mL in lysis buffer) for 2 h at 4 C. The complexes (biotinylated hormone/receptor) formed in both experimental conditions were precipitated with streptavidin beads. For the endoglycosidase H (Endo-H) resistance test, the deglycosylation was performed using the Boehringer kit; the samples were denatured as prescribed by the manufacturer and incubated for 1 h at 37 C with or without Endo-H in reaction buffer. The purified proteins were then denatured once again, electrophoresed on a 7.5% polyacrylamide-SDS gel, transferred to a nitrocellulose membrane, and probed with 5 µg/mL of the mAb263 GHR antibody (Biogenesis, Bournemouth, UK). Detection was performed with the ECL kit (New England Nuclear, Boston, MA).
Immunofluorescence assays
293 cells were plated on Lab-Tek slides (Nalge Nunc International, Naperville, IL; 4 x 105 cells/1-cm2 well) and transfected with either 1 µg WT hGHR or the mutant receptor cDNA constructs. Forty-eight hours after transfection, the cells were washed twice with PBS, fixed with 3% paraformaldehyde, and permeabilized with Triton as described previously (37) or were left nonpermeabilized. Cells were then stained with 5 µg/mL anti-GHR monoclonal antibody (mAb263, Biogenesis). Double labeling was performed with a polyclonal antiendoplasmic reticulum (anti-ER) antibody (pAb203, gift from Daniel Louvard). After washing, the secondary antibodies [antimouse antibody conjugated with fluorescein isothiocyanate (FITC) and/or antirabbit antibody conjugated with rhodamine, both from Organon Teknika, Turnhout, Belgium] were added. Slides were analyzed on a confocal microscope.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Complete screening of all 9 coding exons of the GHR gene by DGGE revealed a single abnormality, suggesting a sequence alteration in only 1 of the 4 patients. A heterozygous pattern was evident for PCR products of exon 9 from patient 3. Subsequent DNA sequencing revealed that patient 3 was heterozygous for a deletion of 13 nucleotides in exon 9, predicted to result in 14 missense codons, followed by a termination codon in the intracellular domain of the receptor. Family studies revealed that the father of patient 3 also carried the exon 9 deletion. The consequences of this frameshift mutation leading to a stop codon will be reported elsewhere. No DGGE alteration was detected to account for the other mutant allele in patient 3.
As the Meltmap computer program (38) predicted that the exon 6 amplimer
has two melting domains, we reasoned that a mutation in the higher
temperature melting domain could have been missed by DGGE analysis.
Therefore, we carried out direct DNA sequencing of exon 6 amplimers
including the intron-exon boundaries. This strategy revealed mutations
in all four patients (Fig. 1
). Patient 1
was homozygous for a CAA to CCA substitution in codon 154 (Q154P),
patient 2 was homozygous for a GAT to CAT substitution in codon 152
(D152H), patient 3 was heterozygous for a ATC to ACC substitution in
codon 153 (I153T), and patient 4 was homozygous for a GTG to GGG
mutation of codon 155 (V155G).
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). Each of the 4 identified exon
6 missense mutations alters a restriction enzyme site generating
altered fragment migration patterns on agarose gel electrophoresis
(Table 1). PCR amplification followed by restriction digest and agarose
gel analysis confirmed the presence of each of these mutations in the
appropriate proband and indicated that none of the 110 control GHR
alleles tested carried a D152H, I153T, Q154P, or V155G mutation.
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. Binding characteristics of the mutant receptors expressed in 293 cells
We previously reproduced the D152H mutation, which was found in
two other patients from unrelated families. We showed that this
substitution induces combined defects in receptor expression,
dimerization, and signaling, as judged on the defective activation of a
STAT5 reporter gene (29). Impaired dimerization of GHBP produced from a
cDNA with the same mutation was also shown (28). To analyze the
functional consequences of the missense mutations found in the three
other patients, the mutations were reproduced in the cDNA of the human
GHR, and plasmids encoding the WT GHR and the three mutant receptors
were transfected into human 293 cells. We first measured binding of
[125I]hGH at the cell surface of transfected cells (Fig. 2A). Specific binding to cells expressing
the three mutant receptors was about 7%, 2%, and 3%, respectively,
of the value measured in cells transfected with the WT GHR cDNA. As
these binding defects could be due to a decrease in the total number of
receptors synthesized or to an abnormal routing of the mutant receptors
to the plasma membrane, we performed binding experiments with total
particulate fractions prepared from transfected cells (Fig. 2B).
Surprisingly, the three mutations seemed to differentially affect the
binding activity of the receptor. A very low binding level (<5%) was
detected in membranes from cells transfected with Q154P mutant cDNA
regardless of the amount of cDNA transfected. This was consistent with
the low binding detected at the surface of cells expressing this
mutant. In contrast, only slight or moderate binding defects were
observed with membrane fractions from cells expressing the I153T and
V155G mutants. Specific binding of [125I]hGH to membrane
fractions (100 µg proteins) from cells expressing the I153T and V155G
hGHRs was about 53% and 26%, respectively, of the specific binding to
membrane fractions from cells expressing the WT GHR construct.
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).
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To visualize the receptors independently of their ability to
bind the hormone, we performed immunofluorescence studies with a
monoclonal antibody recognizing an epitope in the extracellular domain
of the receptor, but not in the region of the mutations. Cells were
fixed and incubated directly or after permeabilization with Triton to
visualize the receptors present at the cell surface (Fig. 3A) and in intracellular compartments
(Fig. 3B). A cell surface fluorescent signal was detected only at the
surface of cells expressing the WT GHR, but not on cells expressing the
I153T, Q154P, or V155G mutant GHR. This result suggested that a very
low number of any of the three mutant receptors was present at the cell
surface. In contrast, comparable levels of immunofluorescence were
observed in intracellular compartments of cells expressing the WT GHR
or the three mutant receptors, suggesting that the receptor proteins
were synthesized in comparable amounts.
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). Although the majority
of the WT GHR signals were colocalized with the ER labeling, additional
labeling could be detected (visualized in green) that might correspond
to Golgi and endocytic compartments in addition to the cell surface
receptors. In contrast, the three mutant GHRs appeared to be entirely
colocalized with the ER marker.
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Western blot analyses were performed to evaluate the sizes
of the WT and the mutant receptors. Transfected cells were incubated
with biotinylated hormone before lysis to selectively label receptors
at the cell surface or after lysis to label all cellular receptors.
Affinity-purified receptors were revealed with the mAb263 monoclonal
antibody (Fig. 5). In cells expressing
the WT GHR (lanes a, b, and c), a broad band ranging from 100–130 kDa
was visualized, suggesting that differently glycosylated hGHR species
were purified. Comparison of the signal corresponding to receptors
located at the cell surface only (lane a) with the signal corresponding
to total cell receptors (lane b) indicated that, as expected (39) (Fig. 3), most of the hGHRs are located in intracellular compartments. Endo-H
digestion of WT receptors resulted in a small shift of the lower
molecular species of the WT GHR, suggesting that the other forms
correspond to receptors that have acquired complex glycosylation in
post-ER compartments. Similar analyses were performed on fractions of
cells expressing the mutant receptors. In the case of cells expressing
the I153T or the V155G mutant, a 100-kDa mol wt band corresponding to
the receptor was detected with mAb263. However, in either case, the
intensities of the bands corresponding to receptors at the cell surface
(lanes d and j) or total receptors (lanes e, f, k, and l) were lower
than those observed with corresponding fractions from cells transfected
with the WT GHR cDNA. In the cells expressing the Q154P mutant, no
signal could reliably be detected by Western blot analysis (lanes g, h,
and i). These results are consistent with the various binding defects
that we measured by [125I]hGH assays. When total cell
mutant receptors (I153T or V155G) were submitted to Endo-H digestion,
most of the receptors appeared to be Endo-H sensitive as demonstrated
by the shift of the bands in lanes f and l, suggesting that these
mutant receptors did not acquire complex glycosylation and were
essentially trapped in the ER.
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The three novel mutations that we identified are located
in the Bß strand of the membrane proximal extracellular ß-sheet of
the hGHR (10). Figure 6 shows alignment
of the sequence of this ß-strand with other members of the cytokine
receptor superfamily (40, 41) and the fibronectin type III domain.
Shaded residues are the topo-hydrophobic amino acids that are known to
be conserved in the cytokine receptor family and probably play a
structural role in hydrophobic core stabilization. A more complete
alignment of 52 domains of Igs and cytokine receptors shows that the
equivalent of positions 153 and 155 in hGHR are occupied by hydrophobic
residues in 92% and 94% of cases, respectively (Poupon A. and Mormon,
J. P., unpublished observations).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patient 1, who was homozygous for Q154P, was the first North American individual with the typical clinical picture of LS and the first in whom GH resistance was demonstrated. Substitution of glutamine by proline at position 154 replaces an uncharged polar side-chain with a nonpolar one, resulting in severe consequences on the binding ability of the mutant receptor expressed in transfected cells. No specific [125I]hGH binding to cell surface or total particulate fractions from cells expressing this mutant could be detected even when high amounts of cDNA were transfected in the 293 overexpression system. Western blot detection was not possible for this mutant, as the mAb263 antibody is not efficient enough in immunoprecipitation, and this mutant was unable to bind biotinylated GH. Immunocytochemistry experiments with mAb263 and colocalization experiments with the anti-ER antibody, however, suggested that the mutant protein was expressed but remained sequestered in the ER compartment.
The moderately lower binding affinity of the I153T and V155G mutants cannot be the only explanation of the extreme GH resistance of the LS patients. Several lines of evidence demonstrated an abnormal intracellular trafficking for these mutants: 1) very low GH binding level was measured at the cell surface, but only a 2-fold decrease in binding to total particulate fractions was found in cells expressing the I153T mutant; 2) the abnormal cell surface routing of these mutants, suggested by the binding experiments, was confirmed by immunocytochemical studies and double labeling experiments with ER; 3) the almost complete digestion with Endo-H of the mutant receptors supports the hypothesis that most of I153T and V155G mutant receptors acquired coreglycosylation only, whereas the decreased intensity of the bands that we detected on Western blots reflected the binding defects.
We cannot exclude for these mutants other functional consequences, such as those found for the D152H mutation. This mutation was identified in the homozygous state in patient 2, but had previously been reported in two LS individuals (28). Although the patients are not known to be related, the mutant alleles share the same haplotype and were detected in individuals of the same ethnic background. We had previously analyzed this mutation and had found that the major abnormalities were partially impaired dimerization and defective signal transduction, whereas the expression level of the mutant receptor was decreased 2-fold compared to the expression level obtained in cells transfected with WT GHR cDNA (29).
It was not possible to assay or to get precise information about the GHBP levels in patients 1 (Q154P) and 4 (V155G). However, the very low surface expression of the receptors with the three new mutations was associated with severely reduced generation of GHBP in cells transfected with the cDNA of the mutants (data not shown). Thus, we could not study the potential formation of GHBP dimers or the activation of reporter genes by mutant receptors.
In patient 3, the mutation I153T was present on one allele only, and on the other allele, a frameshift mutation in exon 9 hGHR was identified. This latter mutation results in an abnormal receptor missing most of the cytoplasmic domain. Thus, even if in this patient the I153T substitution only partially impairs receptor function, the coexistence of the two mutations results in complete GH resistance.
Crystallographic resolution of the GH complexed to two GHBP molecules (10) indicates that the extracellular region of the GHR is folded into two ß-sheets, each composed of seven antiparallel ß-strands, in a similar manner to the globular fibronectin type III domain (an Ig-like domain) (42, 43). The Ile153, Gln154, and Val155 residues are located in the middle of the Bß-strand at the surface of the C-terminal domain of the GHBP (membrane proximal domain). Superposition of this domain with other domains of cytokine receptors of the superfamily and with several related fibronectin III domains shows that the Ile153 and Val155 residues are conserved hydrophobic residues involved in the hydrophobic core stabilization (topohydrophobic residues) (41). Thus, mutations of these amino acids in a hydrophilic threonine and a side-chainless glycine, respectively, probably locally disturb the hydrophobic core formation and stabilization. In opposite, the solvent-exposed Gln154 residue points to the other receptor when the complex is formed. However, it does not participate to the dimerization process in terms of hydrogen bonds (10, 29), but mutation of Gln154 into proline probably disturbs the local conformation as prolines are known to be ß-breakers (44).
Most of the missense mutations of the GHR identified in patients with LS or idiopathic short stature are located in the N-terminal ß-sheet; they directly affect the GH-binding sites without disturbing the expression and folding of the receptor (e.g. E44K) (45). In contrast, the few missense mutations of the membrane proximal domain previously described seem to more structurally affect the GHR. For instance, the R211H mutation seriously impairs GHR expression (45); the R161C GHR is potentially misfolded because of the extra cysteine; the E224D mutation in GHR (45) and the S199I mutation in chick GHR (46) probably involve abnormal subcellular localization because they take place in the very conserved WSxWS-like motif YGEFS (47). Lastly, the substitution of aspartate 152 residue into a histidine (D152H) seems to disturb the dimerization and functionally affect GH signal transduction while the GH binding affinity remains intact (28, 29). The three new mutations we identified are located close to this dimerization region, but we show that they essentially affect different steps in receptor trafficking and function, mainly binding for the Q154P mutant and intracellular transport pathways for the I153T and V155G mutants.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Medicine, University of Iowa
Hospitals, Iowa City, Iowa 52242.
Received May 19, 1998.
Revised September 1, 1998.
Accepted September 14, 1998.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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 M. Maamra, J. Finidori, S. Von Laue, S. Simon, S. Justice, J. Webster, S. Dower, and R. Ross Studies with a Growth Hormone Antagonist and Dual-fluorescent Confocal Microscopy Demonstrate that the Full-length Human Growth Hormone Receptor, but Not the Truncated Isoform, Is Very Rapidly Internalized Independent of Jak2-Stat5 Signaling J. Biol. Chem., May 21, 1999; 274(21): 14791 - 14798. [Abstract] [Full Text] [PDF]
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