Grb10 is a protein that binds to the intracellular domains of activated tyrosine kinase receptors, including insulin-like growth factor (IGF-I) and insulin receptors. This occurs through the interaction of two C-terminal Grb10 motifs (BPS and Src homology domains) with receptor phosphotyrosine residues. Published data from transfection/overexpression studies support both positive and negative regulatory effects of Grb10, thus leaving its physiological role unclear. Because Grb10 has the structure of an adapter protein, the objective of this study was to determine whether Grb10 links other proteins to IGF-I receptors and thus modulates IGF-I signaling. Using yeast two-hybrid screening, the N terminus of Grb10 was shown to interact with two novel proteins, designated GIGYF1 (Grb10 interacting GYF protein 1) and GIGYF2. Mutation analysis indicates that a 17-amino acid sequence in GIGYF1 and GIGYF2, homologous to the GYF domain described previously, binds to tandem proline-rich regions in the N terminus of Grb10. In IGF-I receptor-expressing R؉ fibroblasts, there is detectable binding of a Myc-tagged fragment of GIGYF1 to Grb10 in the basal state. Stimulation with IGF-I results in increased binding of GIGYF1 to Grb10 and transient binding of both Grb10 and GIGYF1 to IGF-I receptors, presumably via the adapter function of Grb10. At later time points, GIGYF1 dissociates, but Grb10 remains linked to IGF-I receptors. Overexpression of the Grb10 binding fragment of GIGYF1 in R؉ cells results in a significant increase in IGF-I-stimulated receptor tyrosine phosphorylation. In conclusion, we have identified two members of a novel protein family, which become transiently linked to IGF-I receptors by the Grb10 adapter protein following IGF-I stimulation. Grb10 and GIGYFs may act cooperatively to regulate receptor signaling.
Obesityis a common problem in western society that is directly linked to several disease processes and is associated with significant morbidity and mortality. Adipocytes -the primary site for energy storage (as triglycerides) and release -were long suspected to have an active role in regulating body weight homeostasis and energy balance. As a result, many studies have focused on finding abnormalities in adipocyte physiology and metabolism. An ever-increasing body of evidence indicates that, in addition to serving as a repository for energy reserves, adipocytes secrete a myriad of factors that comprise a complex network of endocrine, autocrine, and paracrine signals. Very little is known regarding the molecular mechanisms utilized by the adipocyte in regulating the biosynthesis and exocytosis of these secreted products. In order to gain a better understanding of these processes, we have examined the two classical secretory pathways: regulated and constitutive.Using leptin as a model adipocyte-secretory protein, this review focuses primarily on the latter pathway. This includes regulation of leptin synthesis and secretion by insulin and glucocorticoids and, more recently, the finding that the orexigenic neuropeptide, melanin-concentrating hormone (MCH), can stimulate leptin synthesis and secretion. This chapter also incorporates new data describing the partial purification and effect of insulin on leptin-containing vesicles in rat adipocytes. These data indicate that the majority of leptin trafficking occurs via a constitutive secretory pathway and that the primary acute insulin effect on leptin secretion is to increase leptin protein content. In addition, we describe the identification and characterization of the vesicle-associated protein, pantophysin, which may play a multifunctional role in vesicle biogenesis and transport.
Gene expression studies in eosinophilic esophagitis support an immune-mediated etiology associated with differential regulation of inflammatory and epithelial-derived genes. We aimed to further characterize epithelial gene expression alterations in eosinophilic esophagitis and to explore the use of immunohistochemistry to identify these alterations. Esophageal biopsies from pediatric patients with eosinophilic esophagitis before and after therapy with topical steroids (N ¼ 7) were screened by gene expression microarray and results were validated by RT-PCR. A larger group of eosinophilic esophagitis patients (N ¼ 42) was then used to evaluate protein expression by immunohistochemistry compared with reflux patients (N ¼ 15) and normal controls (N ¼ 17). Microarray and RT-PCR studies identified overexpression of ALOX15 and tumor necrosis factor alphainduced factor 6 (TNFAIP6) and underexpression of filaggrin (FLG), SLURP1 and cysteine-rich secretory protein 3 (CRISP3) in eosinophilic esophagitis. Immunohistochemistry for ALOX15 was positive in 95% of eosinophilic esophagitis and negative in all controls, all eosinophilic esophagitis after therapy and all reflux biopsies (Po0.001). TNFAIP6 was positive in 88% of eosinophilic esophagitis samples versus 47% of controls, 29% of eosinophilic esophagitis after therapy and 40% of reflux samples (P ¼ 0.002). Overexpression of both ALOX15 and TNFAIP6 directly correlated with the degree of eosinophilic infiltration. FLG was positive in 88% of controls and 100% of reflux biopsies, but negative in all eosinophilic esophagitis samples, and its expression was regained in 86% of eosinophilic esophagitis after therapy patients (Po0.001). SLURP1 expression was positive in all controls and reflux samples, but only positive in 5% of eosinophilic esophagitis and was re-expressed to 100% positivity in eosinophilic esophagitis after therapy patients (Po0.001). The majority of controls (89%) and reflux biopsies (100%) were positive for CRISP3 while eosinophilic esophagitis before therapy were positive in 14% of samples (Po0.001) with partial recovery after treatment (43%, P ¼ 0.105). This study identified five epithelial-derived markers differentially expressed in eosinophilic esophagitis easily detectable by immunohistochemistry with potential diagnostic utility.
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