Journal of Clinical Endocrinology & Metabolism, Vol 78, 1113-1118, Copyright © 1994 by Endocrine Society

ARTICLES

The stoichiometry of growth hormone-binding protein complexes in human plasma: comparison with cell surface receptors

G Baumann, HB Lowman, M Mercado and JA Wells
Department of Protein Engineering, Genetech, Inc. South San Francisco, California 94080.

The recent demonstration of two independent receptor-binding sites (sites 1 and 2) on human GH (hGH) raises the question of the stoichiometry of circulating GH-binding protein (GH-BP) complexes in human plasma (i.e. is it one hGH per one GHBP or one hGH per two GHBPs?). Previous studies have all assumed 1:1 binding in plasma, based on gel exclusion chromatography and cross-linking data. To address this issue, human plasma was incubated with radioiodinated hGH as well as hGH mutants that had either a Tyr103-->Ala or a Gly120-->Arg substitution in the region of binding site 2. The former mutant retains normal site 2 binding activity even when iodinated; the latter has binding site 2 inactivated. Bound and free hGH were then separated on a Sephadex G-100 column according to a standard protocol for measuring GHBP. In all three cases, more than 90% of the high affinity GH-BP complex eluting from the column was consistent with 1:1 binding. Similar results were obtained when a physiological amount of recombinant or purified natural GHBP was substituted for plasma. However, at supraphysiological concentrations of GHBP, an additional component corresponding to the 2:1 complex eluted from the column; the relative proportions of the 2:1 and 1:1 complexes were dependent on the GHBP concentration. These data suggest that at physiological GHBP levels in plasma, the 1:1 complex predominates, and that small amounts of the 2:1 complex may be difficult to detect because of partial peak overlap with the 1:1 complex, dissociation, and, in whole plasma, coelution with the low affinity GHBP complex. Calculation of the theoretical partition of hGH between 1:1 and 2:1 complexes indicated that at concentrations of GHBP prevailing in plasma (approximately 1 nmol/L), the 1:1 complex predominates, but that at the high receptor concentrations prevailing at the cell surface (60 nmol/L to 6.7 mumol/L, depending on the cell type), virtually all hGH is captured in a 2:1 complex. These findings are consistent with the present and previous experimental data on the size of the circulating high affinity GH-BP complex, as well as with those indicating the importance of GH- induced receptor dimerization for GH action. A functional consequence of the large concentration difference between GHBP in plasma and GH receptors at the cell surface is that the circulating GHBP can serve as a dynamic buffer, modulating bound and free GH and prolonging its half- life, whereas the receptor acts as a dominant force in unidirectional capture of GH.(ABSTRACT TRUNCATED AT 400 WORDS)

This article has been cited by other articles:

				J. Fuglsang, P. Sandager, N. Moller, S. Fisker, H. Orskov, and P. Ovesen Kinetics and secretion of placental growth hormone around parturition. Eur. J. Endocrinol., March 1, 2006; 154(3): 449 - 457. [Abstract] [Full Text] [PDF]

				R. J. M. Ross, K. C. Leung, M. Maamra, W. Bennett, N. Doyle, M. J. Waters, and K. K. Y. Ho Binding and Functional Studies with the Growth Hormone Receptor Antagonist, B2036-PEG (Pegvisomant), Reveal Effects of Pegylation and Evidence That It Binds to a Receptor Dimer J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1716 - 1723. [Abstract] [Full Text]

				G. P. Frick, L.-R. Tai, W. R. Baumbach, and H. M. Goodman Tissue Distribution, Turnover, and Glycosylation of the Long and Short Growth Hormone Receptor Isoforms in Rat Tissues Endocrinology, June 1, 1998; 139(6): 2824 - 2830. [Abstract] [Full Text] [PDF]

				M. Wada, H. Uchida, M. Ikeda, B. Tsunekawa, N. Naito, S. Banba, E. Tanaka, Y. Hashimoto, and M. Honjo The 20-Kilodalton (kDa) Human Growth Hormone (hGH) Differs from the 22-kDa hGH in the Complex Formation with Cell Surface hGH Receptor and hGH-Binding Protein Circulating in Human Plasma Mol. Endocrinol., January 1, 1998; 12(1): 146 - 156. [Abstract] [Full Text] [PDF]

				M. Tzanela, C. Wagner, and G. Shaffer Tannenbaum Recombinant Human Growth Hormone-Binding Protein Fails to Enhance the in Vivo Bioactivity of Human Growth Hormone in Normal Rats Endocrinology, December 1, 1997; 138(12): 5316 - 5324. [Abstract] [Full Text] [PDF]

				A. Di Marco, I. Gloaguen, A. Demartis, I. Saggio, R. Graziani, G. Paonessa, and R. Laufer Agonistic and Antagonistic Variants of Ciliary Neurotrophic Factor (CNTF) Reveal Functional Differences between Membrane-bound and Soluble CNTF alpha -Receptor J. Biol. Chem., September 12, 1997; 272(37): 23069 - 23075. [Abstract] [Full Text] [PDF]

				M. Sundstrom, T. Lundqvist, J. Rodin, LutzB. Giebel, D. Milligan, and G. Norstedt Crystal Structure of an Antagonist Mutant of Human Growth Hormone, G120R, in Complex with Its Receptor at 2.9AResolution J. Biol. Chem., December 13, 1996; 271(50): 32197 - 32203. [Abstract] [Full Text] [PDF]

				D. Pennica, K. J. Shaw, T. A. Swanson, M. W. Moore, D. L. Shelton, K. A. Zioncheck, A. Rosenthal, T. Taga, N. F. Paoni, and W. I. Wood Cardiotrophin-1 J. Biol. Chem., May 5, 1995; 270(18): 10915 - 10922. [Abstract] [Full Text] [PDF]