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

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)