Functional expression of an ovine growth hormone receptor in transfected Chinese hamster ovary cells.
Structural heterogeneity has been demonstrated for growth hormone (GH) receptors from a number of species, and both high and low affinity art receptors have been characterised by ligand binding studies. In the present study, we have transfected Chinese hamster ovary (CHO-K1) cells with a cDNA clone encoding a full-length transmembrane ovine (o) GH receptor, under the regulatory control of the human metallothionein IIA promoter. A stably transfected cell line was established (GHR9.5) which expresses on the cell surface a single class of receptor which binds 220,000 [125I]oGH molecules at high affinity (Kd = 0.30 nM) which is comparable to the affinity established for endogenous oGH receptors in postnatal sheep liver microsomes (Kd = 0.27 nM, Freemark et al. (1987) Endocrinology 120, 1865-1872). The expressed receptor also binds ovine placental lactogen (oPL, 205,000 binding sites per cell) with high affinity (Kd = 0.76 nM). The presence of two species of oGH receptor was detected in GHR9.5 cells using affinity cross-linking analysis (M(r) 148,000 and M(r) 73,000) and given that the oGH receptor cDNA codes for a non-glycosylated receptor of M(r) 69,914, it is likely that these cross-linked species correspond to homodimeric and monomeric forms of the oGH receptor, each binding to a single molecule of GH. Parallel cross-linking studies with sheep liver microsomes also demonstrated two oGH receptor species (M(r) 133,000 and M(r) 58,000), the difference in relative molecular weights between the transfected and endogenous receptors presumably resulting from tissue-specific post-translational modifications. In the presence of oGH, the GHR9.5 cells respond by increasing total cellular protein synthesis by 27% relative to non-GH-exposed GHR9.5 cells, indicating the functionality of the expressed receptor. We also demonstrate unequivocally that oPL, through a specific interaction with the transfected oGH receptor, is able to mediate a similar cellular response (38% protein synthesis induction). Responsiveness to oGH and oPL in the GHR9.5 cells is dependent on serum starvation prior to oGH exposure and occurs only with prolonged exposure (greater than 2 h) to oGH. This cellular stimulation occurs independently of c-fos transcription which has previously been shown to be one of the earliest events associated with GH action in tissues expressing endogenous GH receptors (Doglio et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1148-1152; Slootweg et al. (1990) J. Mol. Endocrinol. 4, 265-274).
Fiddes
R J
RJ
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Brandon
M R
MR
Adams
T E
TE
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology; D008970:Molecular Weight; D011388:Prolactin / Q000494:pharmacology; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D011986:Receptors, Somatotropin / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012756:Sheep; D014162:Transfection
D016760:Proto-Oncogene Proteins c-fos; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D011388:Prolactin; D013006:Growth Hormone; D004247:DNA