Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.     

De novo mutations of the growth hormone gene: an important cause of congenital isolated growth hormone deficiency?

OBJECTIVES: To determine the epidemiological features of injuries associated with fireworks. DESIGN: A retrospective study of reported cases. SUBJECTS: Subjects were those who attended selected Victorian hospital emergency departments (n = 17) and those admitted for firework related injuries (n = 16). RESULTS: The mean (SD) age of attenders at emergency department between January 1988 and June 1996, was 8.9 (6.2) years and most (88%) were under 18 years of age. Males accounted for 71% of the cases. The most common anatomical sites and types of injury were head (47%) and burns (88%), respectively. About 53% of the injuries were caused by firecrackers, the remainder by sparklers and penny bangers. Among those admitted to hospital between July 1987 and June 1996, the mean (SD) age was 22.9 (14.8) years and 50% were under 18 years of age. Males accounted for 87% of the cases. There was a significant difference in mean age between those admitted and not admitted to hospital, the former being significantly older. CONCLUSIONS: Although relatively rare, injuries from fireworks still occur in Victoria after legislative restrictions on their sale in 1985. Consequently, there is a potential risk for injuries among children, particularly from firecrackers. More enforcement of the regulations, education, and parental supervision are needed to prevent injuries from fireworks.     

Constitutive activation of the TSH receptor by spontaneous mutations affecting the N-terminal extracellular domain

Activating mutations of the TSH receptor gene have been found in toxic adenomas and hereditary toxic thyroid hyperplasia. Up to now, all mutations have been located in the serpentine portion of the receptor. We now describe two additional mutations affecting Ser-281 (Ser-281-Thr and Ser-281-Asn) in the ectodomain of the receptor. After transfection in COS cells, both mutants displayed increased constitutive activity for cAMP generation despite expression at a lower level than the wild type. The mutants were responsive to TSH. The present results are compatible with a model in which the activity of the unliganded receptor is kept at a low level by an inhibitory interaction between the N-terminal domain and the serpentine portion of the receptor.     

An immobilised peptide array identifies antibodies to a discontinuous epitope in the extracellular domain of the bovine growth hormone receptor

Using an array of overlapping decapeptides representing the extracellular domain of the bovine (b) growth-hormone receptor (GHR) we have mapped the continuous, dominant epitopes defined by five rabbit and one guinea pig polyclonal antisera to recombinant bovine growth-hormone-binding protein (rbGHBP). We report that six major epitopes are identified by these antisera and that these largely occur in areas of non-ordered secondary structure, although there is some contribution from the extensive beta-sheet structure of GHBP. Similar to our previously described studies for growth hormone (GH), we have again found slight differences between animals in the exact location of these epitopes. Using peptide-affinity chromatography we have isolated a population of antibodies reactive with epitope 1 (the N-terminal epitope:GHBP residues 21-38). Analysis of these antibodies by further peptide affinity chromatography and competitive radioimmunoassay experiments indicated cross-reactivity of epitope-1-specific antibodies with epitope 4 (in the interdomain hinge region of the GHBP:residues 111-126). We suggest that, although separate in the primary structure of the molecule, the tertiary fold exhibited by GHBP may bring into close proximity areas of sequence representing epitope 1 and epitope 4 such that they represent a conformational epitope. Under these conditions our experiments indicate that peptides 1 and 4 may represent partial functional epitopes for this antibody population and consequently demonstrate that this approach may be useful in describing discontinuous epitopes.     

Expression of growth hormone secretagogue-receptors by growth hormone-releasing hormone neurons in the mediobasal hypothalamus

A novel class of synthetic compounds, termed GH-secretagogues (GHSs), have been shown to be potent stimulators of GH release, although their mechanism of action and functional significance remains obscure. The recent cloning of the rat GHS receptor (GHS-R) permitted the identification of numerous sites of expression of GHS-R in brain, but nothing is yet known about the cell types that express this receptor. We performed dual chromogenic and autoradiographic in situ hybridization to test the hypothesis that GHRH neurons in the hypothalamus coexpress GHS-R mRNA. GHS-R-hybridizing cells showed extensive overlap with GHRH-expressing neurons in both the arcuate (Arc) and ventromedial (VMN) hypothalamic nuclei. Quantification of the double-labeled cells revealed that approximately 27% of GHRH-hybridizing neurons in the Arc, and 22% of those in the VMN, expressed the GHS-R gene. These studies are the first to colocalize the GHS-R to any neurochemical cell type in rat brain. The results provide evidence that the GHSs may directly modulate GHRH release, and thereby stimulate GH secretion, through interaction with the GHS-R on hypothalamic GHRH mRNA-containing neurons.     

Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.     

Patterns of ovarian growth and development in cattle with a growth hormone receptor deficiency

Nutritionally induced changes in growth hormone (GH) and IGF-I are associated with decreased ovarian function and may partially explain infertility and anestrus in undernourished cattle. The reproductive importance of GH and IGF-I was tested in cattle with a GH receptor deficiency (GHRD) that have reduced blood IGF-I. Blood was collected daily for plasma, and ovaries were examined daily by ultrasonography for 3 wk during an estrous cycle (estrus = d 0) in GHRD (n = 8) and control (n = 8) cattle. On d 18, blood samples were collected every 10 min for 6 h to measure LH. The GHRD cattle had fewer small antral ovarian follicles (2 to 5 mm, P < .01). After estrous cycle d 5, the first-wave dominant follicle stopped growing in GHRD but continued growing in controls (P < .001). Size of the CL was equivalent for GHRD and controls until d 5, after which CL development slowed in GHRD (P < .01). Likewise, plasma progesterone concentrations were less in GHRD (P < .001). During the luteal phase, GHRD cattle failed to develop follicles greater than 10 mm in diameter (endocrine status x day, P < .05). Size and rate of growth of preovulatory follicles, plasma estradiol, plasma FSH, and plasma LH (d 18 bleed) were similar in GHRD and controls. In conclusion, an important role for GH, GH receptor, and IGF-I in ovarian function was supported because GHRD cattle had distinctly different patterns of ovarian development compared with control cattle.     

Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: Dwarfism of Sindh

We report, in detail, a new form of familial dwarfism, including its phenotypic features, hormonal profile, and molecular basis. Following a newspaper report of severe dwarfism in two villages in the province of Sindh, Pakistan, we organized an expedition to study its clinical, genetic, and molecular characteristics. We identified 18 dwarfs (15 male, 3 female), all members of a consanguineous kindred, ranging in age from newborn to 28 yr. Mean height was 7.2 SD below the norm, with mean adult heights of 130 cm for males and 113.5 cm for females. Body proportions and habitus were normal; but head circumference was 4.1 SD, and blood pressure approximately 3 SD below the norm. There was no dysmorphism, no microphallus, and no history of hypoglycemia. Serum GH did not respond to provocative stimuli (GHRH, L-dopa, or clonidine). Insulin-like growth factor I (IGF-I) and IGF-binding protein 3 were low (5.2 +/- 2.0 ng/mL and 0.42 +/- 0.13 microg/mL, respectively; mean +/- SD) but rose normally with GH treatment. One affected, dwarfed couple had a son, demonstrating fertility in both sexes. Clinical and endocrinological evidence suggested isolated GH deficiency with a recessive inheritance pattern. The GH-N gene was found to be intact. Linkage analysis of microsatellite chromosomal markers near other candidate genes yielded a high LOD score (6.26) for the GHRH receptor (GHRH-R) locus. DNA sequencing revealed a nonsense mutation (Glu50-->Stop) in the extracellular domain of the GHRH-R. This mutation predicts a severely truncated GHRH-R; it is identical to that recently reported in four patients from two other families. Inheritance is autosomal recessive (chromosome 7p) with a high degree of penetrance. Relatives heterozygous for the mutation had moderately decreased IGF-I levels and slightly blunted GH responses to GHRH and L-dopa, but they showed only minimal or no height deficit. This syndrome represents the human homologue of the little (lit/lit) mouse and closely resembles its phenotype. It demonstrates the absolute requirement of GHRH signaling for pituitary GH secretion and postnatal growth in humans, and its relatively minor (but discernible) biological importance in extrapituitary sites. The syndrome is distinct from other forms of GH deficiency with respect to microcephaly, asymptomatic hypotension, and absence of features such as facial dysplasia, significant truncal obesity, microphallus, or hypoglycemia. Its discovery raises the possibility of milder mutations in the GHRH-R gene as potential causes for partial GH insufficiency and idiopathic short stature.     

Effects of adrenalectomy and glucocorticoid receptor antagonist, RU38486, on pituitary growth hormone-releasing hormone receptor gene expression in rats

We examined the effects of adrenalectomy and a glucocorticoid receptor antagonist, RU38486, on pituitary GH-releasing hormone (GRH) receptor gene expression in rats. GRH receptor mRNA levels were significantly decreased in adrenalectomized rats and replacement of dexamethasone reversed the decrease to normal. GH secretion was inhibited by adrenalectomy, whereas dexamethasone replacement failed to restore the impaired GH secretion. A high dose of RU38486 had an agonistic effect on GRH receptor mRNA levels. These results suggest that endogenous glucocorticoid is necessary for normal expression of pituitary GRH receptor mRNA in rats.     

Expression of luteinizing hormone-releasing hormone and its receptor in porcine immune tissues

Luteinizing hormone-releasing hormone (LHRH) is the primary regulator of pituitary LH release. However, LHRH has also been identified in extrahypothalamic sites including immune tissues. Accordingly, immunomodulatory properties for LHRH have been suggested. We wanted to determine whether LHRH and its receptor are produced by immune tissues in the pig. First, a cDNA was cloned and sequenced from the porcine hypothalamus that showed 87.5% homology with the human LHRH gene. Internal primers were identified from this sequence for amplifying a 268 bp product by PCR. In addition to the hypothalamus, PCR products reflecting LHRH mRNA were amplified in porcine spleen, thymus, and peripheral blood lymphocyte (PBL) cDNA. LHRH mRNA was not detected in liver, cerebral cortex, or pituitary tissue samples. Primers were designed to amplify a 360 bp fragment of LHRH-receptor cDNA. PCR products reflecting LHRH-receptor mRNA were amplified in pig hypothalamus, pituitary, thymus, spleen and PBL cDNA samples. No such products were amplified in cortex and liver samples. In summary, we report the sequence of a cDNA coding for LHRH and Gonadotropin-RH associated peptide (GAP) in the pig hypothalamus. Additionally, we provide evidence that LHRH and its receptor are synthesized in porcine immune tissues. This leads us to speculate that LHRH may have local, immunomodulatory functions in pigs.     

Disturbed release of growth hormone in mature dogs: a comparison with congenital growth hormone deficiency

An acquired defect in growth hormone secretion in mature dogs has been associated with some forms of generalised alopecia. In an attempt to elucidate the pathogenesis of the disturbance in growth hormone release, the plasma concentrations of growth hormone and insulin-like growth factor I (IGF-I) were measured in two seven-year-old poodles with alopecia and, for comparison, in two young German sheperd dogs with congenital hyposomatotropism (pituitary dwarfism). In the poodles the basal concentrations of growth hormone were low, although often above the detection limit of the assay. The concentrations of IGF-I were in the reference range for healthy poodles. No growth hormone could be detected in the plasma of the German sheperd dogs and the concentrations of IGF-I were very low. Stimulation with clonidine and growth hormone releasing hormone (GHRH) before and after repeated injections of GHRH did not result in significant increases in growth hormone concentrations in plasma. The concentrations of growth hormone in the poodles fluctuated at low levels during the test period. In the German sheperd dogs the levels of growth hormone remained unmeasurable during the stimulation tests. It was concluded that in the two poodles the basal concentrations of growth hormone were sufficient to maintain normal IGF-I concentrations, and thus the release of growth hormone was considered appropriate. Based upon measurements of urinary corticoids and a review of the literature it is suggested that the lack of a growth hormone response to stimulation was due to the enhanced release of somatostatin as a result of mild and fluctuating hyperadrenocorticism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal intelligence with severe insulin-like growth factor I deficiency due to growth hormone receptor deficiency: a controlled study in a genetically homogeneous population

Superior school performance was reported for 52 Ecuadorian probands with severe deficiency of insulin-like growth factor I (IGF-I) due to GH receptor deficiency (GHRD) resulting from homozygosity for the E180 splice mutation of the GHR. In contrast, subnormal intelligence was reported in a study of 18 genetically heterogeneous Israeli patients, attributed to frequent hypoglycemia or IGF-I dependence of brain development. This study is the first controlled evaluation of the intellectual ability of patients with GHRD. We compared the intelligence of 18 patients of school age (mean +/- SD age, 11.5 +/- 2.8 yr), 42 of their relatives (11.5 +/- 2.8 yr), and 28 community controls (10.0 +/- 0.8 yr), using a battery of intelligence tests that have been validated in cross-cultural research, designed to minimize the effects of physical size, motor coordination, and cultural background. Because all patients had the same GHR mutation, for which the carrier state could be determined, this study also investigated whether heterozygosity for mutation of the GHR among unaffected relatives is associated with intelligence. The intellectual ability of the patients with GHRD was not significantly different from that of their relatives (P > 0.05) on the psychometric tests of intelligence and was comparable to that of the community controls on the chronometric tests. Homozygosity or heterozygosity for the mutation in the GHR gene common to Ecuadorian patients was unrelated to intelligence (P > 0.05). These results indicate that the gene defect causing GHRD is not related to intelligence in the Ecuadorian population. They also indicate that GH-induced IGF-I production is not required for normal brain growth in utero or for postnatal intellectual development.     

Mapping of the sites for ligand binding and receptor dimerization at the extracellular domain of the vascular endothelial growth factor receptor FLT-1

The vascular endothelial growth factor (VEGF) receptor FLT-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind 125I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.     

Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.     

Growth hormone-insulin-like growth factor-I axis in adult insulin-dependent diabetic patients: evidence for central hypersensitivity to growth hormone-releasing hormone and peripheral resistance to growth hormone

The aim of this study was to assess the GH-IGFI axis, GH receptor availability, as reflected by the levels of GH-BP, and the amount of GH-dependent IGFBP-3 in adult IDDM patients with different degrees of metabolic control. Thus, 10 adult well-controlled IDDMs (HbA1 7.8 +/- 0.4%), 10 adult non-ketotic poorly controlled IDDMs (HbA1 13.3 +/- 7%) and 14 sex- and age-matched healthy controls were subjected to two intravenous GH-RH stimulation tests with 0.1 and 1.0 microg/kg body weight respectively, and a plasma IGF-1 generation test induced by the administration of hGH. Poorly controlled IDDM patients exhibited an exaggerated GH response to 1.0 microg/kg of GH-RH when compared to healthy control subjects. Low fasting plasma IGF-1 levels and a blunted IGF-1 response to exogenously administered hGH were also found in poorly controlled IDDMs when compared to the healthy control group. GH-BP levels were significantly lower in IDDMs than in normal controls, and correlated positively with the IGF-1 generation capacity after hGH. Serum IGFBP-3 levels measured by RIA were similar in IDDM and control groups. Good glycemic control for 5.7 +/- 0.9 months did not correct the above mentioned abnormalities of the GH-IGF-1 axis. Our findings suggest that IDDM is associated with a diminished availability of GH receptors and synthesis of IGF-1. GH might then increase as a compensatory mechanism, further down-regulating liver GH receptors, and thus perpetuating the initial abnormality.     

Development and maintenance of ear innervation and function: lessons from mutations in mouse and man

We examined mate switching between mated pairs of monogamous convict cichlids as a function of mate quality (size). A mated pair was established in each half of a 284-litre aquarium, an opaque partition separating the two pairs. When the partition was removed, mis-assorted pairs (large males with small females competing with small males with large females) re-sorted themselves such that the larger male and the larger female paired with each other 46% of the time. In contrast, when we exposed initially assorted pairs to each other, large pairs remained intact most of the time and dominated smaller pairs. The pair containing the large male, whether re-sorted or intact, dominated over the other pair and was the only one seen to spawn. Re-sortment resulted both from a preference of males for larger females and of females for larger males, and from the ability of larger individuals to displace their smaller consexual. Small females, however, when paired with a large male, often dominated large females and prevented the large female from mating with the large male. Re-sortment was also influenced by the compatibility of large individuals in their initial pairing situation. Large individuals that had been more compatible with their initial mates were less likely to switch mates. Our results support both the better-option and the incompatibility hypotheses of mate-switching. The availability of more than one breeding site in the aquarium had no effect on the frequency of re-sortment. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.