No abstract

Keratinocyte growth factor (KGF or FGF-7) is a member of the heparin binding fibroblast growth factor (FGF) family and is a paracrine mediator of proliferation and differentiation of a wide variety of epithelial cells. To examine the stoichiometry of complexes formed between KGF and its receptor, we have utilized a soluble variant of the extracellular region of the KGF receptor containing two tandem immunoglobulin-like loops, loops II and III (sKGFR). Ligand-receptor complexes were examined by size exclusion chromatography, light scattering, N-terminal protein sequencing, and sedimentation velocity. In the presence of low-molecular mass heparin ( approximately 3 kDa), we demonstrate the formation of complexes containing two molecules of sKGFR and one molecule of KGF. In the absence of heparin, we were unable to detect any KGF-sKGFR complexes using the above techniques, and additional studies in which sedimentation equilibrium was used show that the binding is very weak (Kd >/= 70 microM). Furthermore, using heparin fragments of defined size, we demonstrate that a heparin octamer or decamer can promote formation of a 2:1 complex, while a hexamer does not. Utilizing the highly purified proteins and defined conditions described in this study, we find that heparin is obligatory for formation of a KGF-sKGFR complex. Finally, 32D cells, which appear to lack low-affinity FGF binding sites, were transfected with a KGFR-erythropoeitin receptor chimera and were found to require heparin to achieve maximal KGF stimulation. Our data are consistent with the previously described concept that cell- or matrix-associated heparan sulfate proteoglycans (HSPGs) and FGF ligands participate in a concerted mechanism that facilitates FGFR dimerization and signal transduction in vivo.

Leptin and the leptin receptor are key players in the regulation of body weight. In an attempt to dissect the molecular mechanism of the Zucker fatty rat leptin receptor mutation (Gln 269 3 Pro) we analyzed the effects of this mutation on leptin receptor signaling and expression in three different expression systems: 1) 32D cells expressing leptin/erythropoietin receptor chimeras, 2) COS-7 cells expressing a leptin receptor short form, and 3) 293 cells expressing soluble receptor forms. To determine if the Gln 269 3 Pro mutation is critical for the observed phenotype, we made a similar Gln 3 Pro mutation at a vicinal residue two amino acids upstream of the fatty mutation to see if it would have similar effects. Incorporation of either of the Gln 3 Pro mutations into wild type receptor forms did not interfere with leptin binding, but it resulted in a signaling-incompetent receptor. In addition, the majority of the mutant receptor protein was localized intracellularly. Our results suggest that the obese phenotype resulting from the Gln 269 3 Pro mutation in the leptin receptor of the Zucker fatty rat may be due not only to a reduced cell surface expression of this form of the leptin receptor, but also to a post-leptin binding malfunction of the receptor that interferes with subsequent signal transduction.

In an increasing number of hematopoietic cytokine receptor HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activa-erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase tion. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late ery-SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglo-throid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in binization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO may enforce mitogenesis. ᭧