Heparin is essential for a single keratinocyte growth factor molecule to bind and form a complex with two molecules of the extracellular domain of its receptor.

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.

Altered cell surface expression and signaling of leptin receptors containing the fatty mutation.

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 (Gln269 --> 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 Gln269 --> Pro mutation is critical for the observed phenotype, we made a similar Gln --> 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 --> 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 Gln269 --> 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.

Epo-induced hemoglobinization of SKT6 cells is mediated by minimal cytoplasmic domains of the Epo or prolactin receptors without modulation of GATA-1 or EKLF.

Interaction of erythropoietin with its type 1 receptor is essential to the development of late erythroid progenitor cells. Through the ectopic expression of receptor mutants in lymphoid and myeloid cell lines, insight has been gained regarding effectors that regulate Epo-induced proliferation. In contrast, effectors that regulate Epo-induced differentiation events (e.g. globin gene expression) are largely undefined. For in vitro studies of this pathway, erythroleukemic SKT6 cell sublines have been isolated which stably and efficiently hemoglobinize in response to Epo. Epo rapidly activated Jak2, STAT5 and detectably STATs 1 and 3, while no effects on GATA-1, EKLF or STAT5 expression were observed. Finally, efficient hemoglobinization of SKT6 cells was shown to be mediated by chimeric receptors comprised of the EGF receptor extracellular domain and truncated cytoplasmic subdomains of either the Epo receptor or the prolactin Nb2 receptor. This work further establishes SKT6 cells as an important model for studies of Epo-stimulated differentiation, and shows that this signaling pathway is promoted by a limited set of membrane-proximal receptor domains and effectors.

Interferon-gamma receptor: mRNA half-life, receptor mass, and abundance on A431 human epidermoid carcinoma cells.

The human interferon-gamma (IFN-gamma) receptor protein and mRNA were identified in the epidermoid carcinoma cell line A431 by radioligand binding and Northern hybridization, respectively. Receptor affinity and receptor number per cell were calculated by Scatchard analysis of saturation binding data. The half-life of the receptor mRNA was determined by actinomycin D inhibition of de novo transcription. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of covalently cross-linked 32P-labeled IFN-gamma and its receptor is consistent with a molecular mass of approximately 99 kD. Radioligand binding analysis revealed 27,000 high affinity (KD = 7.1 x 10(-10) M) receptors per cell. Northern hybridization using an IFN-gamma receptor cDNA probe revealed a single mRNA of 2.2 kb. The receptor mRNA half-life in actinomycin D-treated cells was 4.7 h.