Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms

• Published in: The Journal of biological chemistry Vol. 267; no. 25; pp. 17792 - 17803
• Main Authors: JIANMING XU, NAKAHARA, M, CRABB, J. W, ERGANG SHI, MATUO, Y, FRASER, M, KAN, M, JINZHAO HOU, MCKEEHAN, W. L
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.09.1992
• Subjects: Amino Acid Sequence; Animals; Antibodies; Baculoviridae - genetics; Base Sequence; biochemical characteristics; Biological and medical sciences; Blotting, Western; Cell Line; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; fibroblast growth factor; Fibroblast Growth Factors - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Variation; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; immunological properties; Insecta; isoforms; man; membrane proteins; Models, Molecular; Molecular and cellular biology; Molecular Sequence Data; Peptides - chemical synthesis; Peptides - immunology; Protein Conformation; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Rats; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 2; receptors; Receptors, Cell Surface - genetics; Receptors, Cell Surface - isolation & purification; Receptors, Cell Surface - metabolism; Receptors, Fibroblast Growth Factor; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA Splicing; Sequence Homology, Nucleic Acid; Transfection; Characterization; Human; Fibroblast growth factor; Vertebrata; Molecular form; Membrane receptor; Mammalia; Rat; Rodentia; Spatial structure
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)37114-5

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.