Up-regulation of GH-binding protein by mouse GH in transgenic mice overexpressing GH-releasing hormone.
To study the effects of homologous mouse GH (mGH) on the presence and characteristics of serum GH-binding protein (GHBP) we have used transgenic mice expressing GH-releasing hormone (GHRH) as a model. Chromatographic techniques allowed the characterization of GHBP bioactivity, and immunological techniques were used to determine its concentration and molecular components. Chromatographic separation of labeled human GH or mGH cross-linked to serum GHBPs showed two GH-binding serum fractions in normal as well as in transgenic mice serum. SDS-PAGE of this material revealed a specific band of 66 kDa and another higher molecular weight broad band, which, in the presence of 2-mercapto-ethanol, is converted into the 66 kDa fraction. Since normal mice serum has an mGH concentration of 0. 40+/-0.06 nM and a GHBP concentration of 5.7+/-1.1 nM, while the high-affinity site for mGH has a K(d)</+/-27 nM, only a small percentage (2.9%) of total serum mGH is bound to GHBP in the sera of these mice. In transgenic mice serum, where the mGH concentration is 60 times higher (23+/-2.7 nM), 22.5% of total serum mGH is bound to serum GHBP. These values agree with the experimental data (4+/-2% and 17+/-4% for normal and transgenic mice serum respectively). The concentration of GHBP in GHRH transgenic mice was found to be increased four- to tenfold, depending on the technique used. This increment closely resembles the increased concentration of GHBP in the serum of transgenic bovine GH (bGH) mice, in which peripheral bGH levels are grossly elevated. Our results support the idea that the circulating levels of mGH in normal mouse serum are capable of influencing the levels of GHBP in peripheral circulation in a way similar to that of bGH, in spite of the different affinities of these two hormones. The fact that the up-regulation of GHBP occurs, even though a small percentage of mGH is bound in these animals, strongly suggests the existence of a physiological function for GHBP. These results also question some of the assigned or attributed physiological roles of GHBP, at least in the mouse, since only a negligible percentage of total mGH would be prevented from degradation and/or renal filtration by binding to GHBP. This small percentage of bound mGH also invalidates its role as a reservoir or a buffer of mGH concentration during pulses of GH release or rapid changes of mGH levels. Our results also demonstrate the presence of high molecular weight forms of GH-GHBP complexes that could be dissociated by dilution or in the presence of 2-mercapto-ethanol.
González
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Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Junín 956, (1113) Buenos Aires, Argentina.
Sotelo
A I
AI
Bartke
A
A
Turyn
D
D
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood / Q000187:drug effects*; D002417:Cattle; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D005260:Female; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D015854:Up-Regulation
D002352:Carrier Proteins; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; C051815:somatotropin-binding protein