Functional expression of guinea pig growth hormone receptor and its mutants in mammalian cells.
The cDNA of guinea pig (Cavia porcellus) growth hormone receptor (gpGHR) was cloned using RT-PCR in our laboratory. By sequence alignment, substitutions of amino acids conserved in other mammalian GHRs were found. For example, histidine-168 and tyrosine-332 equivalent to positions 170 and 333 in other mammalian GHRs, which were considered to be necessary for the dimerization of GHR and the specific GH-stimulated functions respectively, were replaced by tyrosine and serine in gpGHR. Here, we report the functional expression of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or Chinese hamster ovary (CHO) cells. It was shown that the COS-7 cells transfected with pcDNA3-gpGHR possessed high affinity to bovine GH [K(a) = 1.3 x10(9) (mol/L)(-1)] and a protein band with molecular weight around 92 kD was detected by anti-mouse GHR monoclonal antibody (mAb263). When CHO cells were transfected with the expression vectors, pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its mutants were expressed and the ligand binding, phosphorylation of JAK2, protein synthesis, and lipogenesis were studied. It was found that the mutation of serine to tyrosine at position 332 greatly increased the GH-stimulated protein synthesis and the phosphorylation of JAK2, while the mutation of tyrosine to histidine at position 168 increased the protein synthesis and decreased the phosphorylation of JAK2 only weakly. However, both mutations decreased the GH-stimulated lipogenesis. Thus, our study provides the experimental evidence that gpGHR may mediate the metabolic actions of GH and the substitutions of some conserved amino acids in gpGHR result in the changes of post-binding signaling.
Liao
Zhi-Yong
ZY
Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.
Zhang
Xin-Na
XN
Jing
Nai-He
NH
Zhu
Shang-Quan
SQ
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D005260:Female; D006168:Guinea Pigs; D009154:Mutation; D011986:Receptors, Somatotropin / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D011986:Receptors, Somatotropin