PCR-based identification of new receptors: molecular cloning of a receptor for fibroblast growth factors.
Transmembrane tyrosine kinases are involved in the control of cell growth and differentiation by extracellular signals. To enable identification of new receptor tyrosine kinases we developed a method that selectively amplifies segments of receptor genes. The method is based on a combination of polymerase chain reaction (PCR) and hybridization screening and it employs three oligonucleotide primers derived from conserved domains of receptor tyrosine kinases. It yields amplification of receptors' genes and appears to ignore cytoplasmic tyrosine kinases. When applied to RNA from 12.5 days post coitum mouse placenta, this methodology resulted in the detection of several putative or established receptors. Molecular cloning of one of these genes, which is identical to the partially characterized bek gene, identified a transmembrane tyrosine kinase with three immunoglobulin-like domains in the extracellular portion, and a cytoplasmic tyrosine kinase sequence. The isolated cDNA shows remarkable homology to the murine flg gene that encodes a receptor for fibroblast growth factors. Indeed, an antibody directed to the carboxy terminus of the deduced bek protein specifically recognized a receptor for acidic and basic fibroblast growth factors in murine hepatoma cells. We therefore expect that the methodology we developed will enable the study of new receptors in hardly accessible biological systems such as early mammalian embryos or stem cells.
Raz
V
V
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Kelman
Z
Z
Avivi
A
A
Neufeld
G
G
Givol
D
D
Yarden
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D005260:Female; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D015723:Gene Library; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010920:Placenta / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; D011956:Receptors, Cell Surface / Q000235:genetics*; D017468:Receptors, Fibroblast Growth Factor; D012689:Sequence Homology, Nucleic Acid
D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D012313:RNA; C496354:Fgfr2 protein, mouse; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2