Functional expression of a mouse growth hormone receptor cDNA in transfected mouse L cells.
A cDNA encoding a full-length mouse (m) growth hormone receptor (GHR) derived from 3T3 F442A cells was amplified by RT-PCR and cloned into a mammalian expression vector. A mouse L cell line (mGHR1.6), which expresses high levels of full-length mGHR, was established. A mGHR-specific mRNA of approx 2.8 kb was found in these cells. Ligand binding studies showed that mGHR 1.6 cells were capable of binding 125I-hGH with a dissociation constant (Kd) of 2.9 +/- 0.13 nM. Scatchard analysis indicated that mGHR1.6 cells had only a single class of mGHR and possessed approx 128,000 GH specific binding sites per cell. Affinity crosslinking studies showed that the recombinant mGHR possessed an apparent molecular mass of 105 kDa. In addition, mGHR1.6 cells responded to growth hormones (GHs) from several species. Two proteins, pp92 and pp95, were found to be tyrosyl phosphorylated following GH treatment. An hGH antagonist, hGH-G120R, inhibited GH-induced phosphorylation of both pp92 and pp95 in a dose-dependent manner. This cell line may be used as an in vitro model in the studies of GH signal transduction and in the screening of GH analogs for biological activity.
Zhou
Y
Y
Department of Biological Sciences, Ohio University, Athens 45701.
Xu
B
B
Wang
X
X
Chen
W Y
WY
Kopchick
J J
JJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D013006:Growth Hormone / Q000378:metabolism*; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000235:genetics*; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D014443:Tyrosine; D013006:Growth Hormone; D012194:RNA-Directed DNA Polymerase