Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.
In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.
Jensen
R A
RA
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Beeler
J F
JF
Heidaran
M A
MA
LaRochelle
W J
WJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D010766:Phosphorylation; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D017479:Receptors, Platelet-Derived Growth Factor