Assignment of the human GRB14 gene to chromosome 2q22-q24 by fluorescence in situ hybridization.
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Adaptor Proteins, Signal Transducing↗
Biomedical subjects
R J Daly
Publications and source records attributed to R J Daly.
Cloning and characterization of GRB14, a novel member of the GRB7 gene family.
Screening of a human breast epithelial cell cDNA library with the tyrosine-phosphorylated C terminus of the epidermal growth factor receptor identified a novel member of the GRB7 gene family, designated GRB14. In addition to a pleckstrin homology domain-containing central region homologous to the Caenorhabditis elegans protein F10E9.6/mig 10 and a C-terminal Src homology 2 (SH2) domain, a conserved N-terminal motif, P(S/A)IPNPFPEL, can now be included as a hallmark of this family. GRB14 mRNA was expressed at high levels in the liver, kidney, pancreas, testis, ovary, heart, and skeletal muscle. Anti-Grb14 antibodies recognized a protein of approximately 58 kDa in a restricted range of human cell lines. Among those of breast cancer origin, GRB14 expression strongly correlated with estrogen receptor positivity, and differential expression was also observed among human prostate cancer cell lines. A GST-Grb14 SH2 domain fusion protein exhibited strong binding to activated platelet-derived growth factor (PDGF) receptors (PDGFRs) in vitro, but association between Grb14 and beta-PDGFRs could not be detected in vivo. In serum-starved cells, Grb14 was phosphorylated on serine residues, which increased with PDGF, but not EGF, treatment. Grb14 is therefore a target for a PDGF-regulated serine kinase, an interaction that does not require PDGFR-Grb14 association.
Adaptor Proteins, Signal Transducing↗
The influence of age, gender and ABO blood group on soluble endothelial cell markers and adhesion molecules.
The influence of age, gender and ABO blood group on soluble adhesion molecules and markers of endothelial function were tested by measurement of levels of von Willebrand factor, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), P-selectin, E-selectin and thrombomodulin in venous blood from healthy subjects. Only VCAM-1 showed a significant multivariate correlation with age (r = 0.1, P = 0.019), and thrombomodulin (P = 0.0001) and E-selectin (P = 0.026) were lower in the women. Significant differences between ABO blood groups were found for von Willebrand factor (P = 0.024), E-selectin (P < 0.001) and thrombomodulin (P < 0.001). As these three are all specific endothelial products, our findings may have implications for vascular biology.
ABO Blood-Group System↗
Self-reported smoking in vascular disease: the need for biochemical confirmation.
The relationship between smoking and vascular disease requires reliable information on the smoking status of patients suffering these disorders. As self-reported smoking status is unreliable and therefore misleading, biochemical markers (such as serum thiocyanate) provide a more accurate guide to smoking status. This study compared the self-reported and biochemical marker data (serum thiocyanate) for two different groups: 215 patients with peripheral vascular disease (PVD) and 124 with ischaemic heart disease (IHD). The object of the comparison was to assess if there was any difference between the two groups with regard to denial of smoking. The PVD group had more smokers by either criterion (verbal 34% in PVD, 15% in IHD, P < 0.001: thiocyanate level > 12.5 micrograms ml-1, 47% in PVD, 34% in IHD, P < 0.05). Of those verbally claiming and biochemically assessed as smokers, 55% of those with IHD and 28% of those with PVD denied using cigarettes. Our data point to differences in verbal reporting of smoking between patients with IHD and PVD. This probably reflects the patient's perception of the dangers of smoking and their diseases and may have clinical consequences for the collection of accurate data on the influence of this risk factor on atherosclerosis.
Biomarkers↗
SH2 domain-containing signaling proteins in human breast cancer.
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Adaptor Proteins, Signal Transducing↗
Progression to steroid autonomy is accompanied by altered sensitivity to growth factors in S115 mouse mammary tumour cells.
Progression to steroid autonomy is a major clinical problem in the treatment of steroid-sensitive tumours. Molecular mechanisms remain unknown but recent hypotheses imply a role for growth factors in this progression. Since S115 + A androgen-responsive mouse mammary tumour cells provide a model system to study this phenomenon in vitro, we have used this model to investigate growth factor gene expression and sensitivity during progression from a steroid sensitive to insensitive state. S115 + A androgen-responsive cells showed a positive proliferative response, morphological response and increased saturation density to various forms of fibroblast growth factor (FGF) and transforming growth factor beta (TGF beta) in both monolayer and suspension culture. A marked synergy was noted, however, between FGF and TGF beta in promoting growth in suspension culture. S115 + A cells possessed mRNA for both acidic FGF (aFGF) and TGF beta 1, both of which were increased by testosterone. Progression to androgen insensitivity was associated with a reversal of growth factor response such that all growth factor responses became generally inhibitory on growth of the unresponsive cells but with a particularly striking synergistic action between FGF and TGF beta 1 on inhibition of both monolayer and suspension growth. Levels of aFGF and TGF beta 1 mRNAs remained low in steroid-insensitive S115-A cells, indicating that loss of response was not associated with any constitutive upregulation of endogenous production of one of these growth factors. The scientific and clinical implications are discussed.
Androgens↗
Exposure of Gambian children to Anopheles gambiae malaria vectors in an irrigated rice production area.
Variation in exposure of children to malaria vectors of the Anopheles gambiae complex was recorded in a Gambian village situated near an irrigated area of rice cultivation. Observations were made in 1987 and 1988 during two dry seasons, when pumped water was used to grow rice, and two rainy seasons, when rice was produced using a combination of irrigated and rainfed paddies. Routine collections of mosquitoes were made from under bednets. Most of these specimens were assumed to have fed on the occupants of the net and thus represented a crude measure of exposure to malaria. Most nets in the village were in good condition, but even these were a poor defence against blood-seeking mosquitoes. Two annual peaks in the numbers of An.gambiae s.l. corresponded with the irrigation of rice paddies in the dry and wet seasons. When there were few vectors in the village the frequency distribution of mosquitoes caught under nets was described best by a Poisson process. When high numbers were present the daily distributions were over-dispersed and fitted a negative binomial model. The spatial distribution of mosquitoes varied between dry and wet seasons and was related to the predominant wind direction at night, suggesting that wind assisted the dispersal of mosquitoes from their breeding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Agriculture↗
Medico-legal aspects of suicide--a clinical perspective.
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Female↗
Interpersonal problem-solving skills training in the treatment of self-poisoning patients.
The present study evaluated the effectiveness of interpersonal problem-solving skills training (IPSST) for the treatment of self-poisoning patients. Thirty-nine self-poisoning patients were assigned randomly either to IPSST or to a control treatment condition (a brief problem-oriented approach). Both conditions were equally effective in reducing the number of presenting problems and in reducing hopelessness levels. However, the IPSST condition was significantly more effective than the control condition as determined by other outcome measures (measures of interpersonal cognitive problem solving, self-rated personal problem-solving ability, perceived ability to cope with ongoing problems, and self-perception). Follow-up studies showed maintenance of IPSST treatment gains at 6 months and a greater reduction of repetition of self-poisoning in the IPSST group at 1 year posttreatment.
Adolescent↗
Activation of the Ras signalling pathway in human breast cancer cells overexpressing erbB-2.
The c-erbB-2 proto-oncogene encodes a receptor tyrosine kinase (RTK) closely related to the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 occurs in approximately 20% of human breast tumours, where increased expression correlates with poor patient prognosis. The EGFR is coupled to the Ras signalling pathway by interaction with the adaptor protein Grb2, and Sos, a Ras GDP-GTP exchange factor. In this study, activation of the erbB-2 receptor and its association with Grb2 and Sos was investigated in breast cancer cell lines which overexpress erbB-2. The receptor was found to be tyrosine phosphorylated in all cell lines in which it is overexpressed. Western blotting of Grb2 and Sos immuneprecipitates from such cells revealed co-precipitation of erbB-2, demonstrating association of the Grb2/Sos complex with erbB-2 in vivo. Furthermore, a fusion protein containing only the SH2 domain of Grb2 bound to erbB-2 immobilized on nitrocellulose, indicating that association with Grb2 is direct and mediated by the SH2 domain of Grb2. The degree of association between the erbB-2 receptor and Grb2 in vivo was related to erbB-2 overexpression, and MAP kinase, which functions downstream from Ras, displayed markedly increased activity in cell lines overexpressing erbB-2. These results demonstrate that erbB-2 is coupled to Ras signalling via the Grb2/Sos complex, and that overexpression of this receptor in breast cancer cells leads to amplification of the Ras signalling pathway.
Adaptor Proteins, Signal Transducing↗
Overexpression of the Grb2 gene in human breast cancer cell lines.
A receptor blotting technique was used to detect SH2 domain containing epidermal growth factor receptor (EGFR) substrates that exhibited differential expression either between normal breast epithelial cells and breast cancer cells or between different human breast cancer cell lines. This identified a 25 kD protein, subsequently identified as Grb2, which was markedly overexpressed in three breast cancer cell lines (MCF-7, MDA-MB-361 and -453) relative to both normal breast epithelial cells and the majority of breast cancer cell lines. Northern blot analysis revealed that 7/19 breast cancer cell lines exhibited more than twofold overexpression of Grb2 mRNA, with overexpression correlating with high expression of erbB receptors. In MCF-7, MDA-MB-361 and -453 cells the overexpression of Grb2 mRNA and protein was accompanied by a small amplification of the Grb2 gene locus. Overexpression of Grb2 correlated with increased complex formation between Grb2 and the hSos-1 Ras GDP-GTP exchange protein. This upregulation of the Ras signalling pathway might modulate the growth factor sensitivity of human breast cancer cells and therefore play a role in tumour progression.
Adaptor Proteins, Signal Transducing↗
The human GRB2 and Drosophila Drk genes can functionally replace the Caenorhabditis elegans cell signaling gene sem-5.
Mutations in the Caenorhabditis elegans gene sem-5 affect cell signaling processes involved in guiding a class of cell migrations and inducing vulval cell fates. The sem-5 sequence encodes a protein comprised almost exclusively of SH2 and SH3 domains (SH, src homology region) that are found together in many signaling proteins and nonreceptor tyrosine kinases. A human protein, GRB2, was identified by its ability to associate with the activated human epidermal growth factor receptor (hEGFR). The GRB2 and Sem-5 proteins share an identical architecture of their SH2 and SH3 domains and 58% amino acid sequence identity. Here we demonstrate that GRB2 and a Drosophila sem-5-like gene Drk can specifically rescue sem-5 mutants. We also show that Sem-5, like GRB2, can bind to the activated hEGFR in vitro. We further correlate the abilities of several mutant variants of GRB2 and Sem-5 to bind to the hEGFR in vitro with their abilities to functionally replace sem-5 in vivo. These data indicate that GRB2 and Drk are functional homologues of Sem-5 and demonstrate the high degree of conservation of both structure and function between signaling systems throughout evolution.
Adaptor Proteins, Signal Transducing↗
Suicide in depressed patients: medico-legal issues.
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Depressive Disorder↗
The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.
A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.
Adaptor Proteins, Signal Transducing↗
SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.
Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
3T3 Cells↗
Autocrine production of insulin-like growth factor II using an inducible expression system results in reduced estrogen sensitivity of MCF-7 human breast cancer cells.
An inducible expression vector, utilizing the metal response elements from the human metallothionein IIA promoter and encoding human prepro-insulin-like growth factor II (IGF-II), was transfected into MCF-7 McG cells, an MCF-7 subline which exhibits an estrogen dependent phenotype in vitro and does not express detectable levels of IGF-II mRNA. Two stably transfected clones, designated MI5 and MI7, which expressed IGF-II mRNA in a Zn2+ regulated manner, were isolated. Clone MI5 did not secrete detectable levels of IGF-II activity, as determined by radioimmunoassay of conditioned medium, but clone MI7 secreted high levels of IGF-II activity in a Zn2+ inducible fashion. Clone MI5 and control clones transfected with the selection plasmid pSV2 neo and the control plasmid pSP65 continued to display an estrogen dependent phenotype in vitro. However, under both anchorage dependent and anchorage independent growth conditions, clone MI7 cells exhibited an estrogen responsive, rather than dependent, phenotype. Moreover, when grown in the presence of inducing concentrations of Zn2+, MI7 cells were either virtually (for anchorage dependent growth) or completely (anchorage independent growth) unresponsive to estradiol. Both the basal growth rate in the absence of metal ions and the Zn2+ induced increases in cell proliferation could be inhibited by the monoclonal antibody alpha-IR3, which blocks the binding site of the IGF-I receptor. The antiestrogens tamoxifen and 4-hydroxytamoxifen were found to enhance the growth stimulation resulting from Zn2+ induced IGF-II production in MI7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Breast Neoplasms↗
Ability of Anopheles gambiae mosquitoes to transmit malaria during the dry and wet seasons in an area of irrigated rice cultivation in The Gambia.
The seasonality of malaria transmission was studied in a Gambian village situated in an area where rice was cultivated. Observations were made during two dry seasons, when pump-fed irrigation was used to grow rice, and in the intervening rainy season, when rice was cultivated using a combination of irrigated and rain-fed paddies. Clinical episodes of malaria were mainly confined to the months during and soon after the rainy season. In the wet season the prevalence of parasitaemia was higher in febrile subjects than in afebrile controls but the reverse applied during the dry seasons. However, the biting rates of Anopheles gambiae complex mosquitoes in the two dry seasons (2.5 and 0.8 bites/child/night respectively) were greater than or similar to that in the rainy season (0.6 bites/child/night). The proportion of human bloodmeals (0.53 vs 0.75) and the survival of mosquitoes (parity rates of 0.41 vs 0.58) were both lower in the dry seasons than in the rains. The low prevalence of morbidity due to malaria in the dry season and the observed fall in the sporozoite rate may therefore have been due to a reduction in the vectorial capacity of the An. gambiae population. However, reduced transmission in the dry season may also have been due to the direct effect of high temperatures on the parasite in the vector.
Age Factors↗