title: Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.
abstract: Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.
authors.affiliation: W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946.
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.forename: M
authors.initials: M
authors.lastname: Nakahara
authors.forename: J W
authors.initials: JW
authors.lastname: Crabb
authors.forename: E
authors.initials: E
authors.lastname: Shi
authors.forename: Y
authors.initials: Y
authors.lastname: Matuo
authors.forename: M
authors.initials: M
authors.lastname: Fraser
authors.forename: M
authors.initials: M
authors.lastname: Kan
authors.forename: J
authors.initials: J
authors.lastname: Hou
authors.forename: W L
authors.initials: WL
authors.lastname: McKeehan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D000091344:Filaggrin Proteins; D014644:Genetic Variation; D006801:Humans; D007313:Insecta; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
chemical_list: D000906:Antibodies; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D005346:Fibroblast Growth Factors; C496353:FGFR2 protein, human; C496356:Fgfr2 protein, rat; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2