Tyrosine 785 is a major determinant of Trk--substrate interaction.
Interaction of the nerve growth factor (NGF) receptor/Trk with cellular substrates was investigated by transient co-overexpression in human 293 fibroblasts using ET-R, a chimeric receptor consisting of the epidermal growth factor receptor (EGF-R) extracellular ligand binding domain and the Trk transmembrane and intracellular signal-generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C gamma (PLC gamma), ras GTPase-activating protein (GAP) and the non-catalytic subunit of phosphatidylinositol-3'-kinase, p85, in a ligand-dependent manner. Deletion of 15 C-terminal amino acids, including tyrosine 785 (Y-785) abrogated receptor and substrate phosphorylation activities. Mutation of Y-785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET-YF phosphorylation of PLC gamma was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine-phosphorylated synthetic C-terminal peptide competitively inhibited Trk cytoplasmic domain association with PLC gamma. Thus, the short C-terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y-785 is a major and selective interaction site for PLC gamma.
Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
A
A
Obermeier
H
H
Halfter
K H
KH
Wiesmüller
G
G
Jung
J
J
Schlessinger
A
A
Ullrich
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA; D066246:ErbB Receptors / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020917:Receptor, trkA; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D004247:DNA; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D020917:Receptor, trkA; D010738:Type C Phospholipases