The Binding between the Stem Regions of Human Growth Hormone (GH) Receptor Compensates for the Weaker Site 1 Binding of 20-kDa Human GH (hGH) than That of 22-kDa hGH

Abstract

Despite the lower site 1 affinity of the 20-kDa human growh hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22-kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K-and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K-and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH.

Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization

“…This observation implied that it is the ECD1-ECD2 stem-stem contact region that plays the major role in stabilizing the ternary complex. Two studies on the effects of stem-stem mutations on cell proliferation have been reported with somewhat different interpretations of which residues contribute most to homodimerization (17,18). Although these biological assays provided a qualitative indication about which stem residues contribute most to homodimerization, it was not possible from the experimental design to discriminate whether a particular mutation produced an effect in context of its role in ECD1, ECD2, or both.…”

Mathematical Model of Human Growth Hormone (hGH)-Stimulated Cell Proliferation Explains the Efficacy of hGH Variants as Receptor Agonists or Antagonists

“…By accounting for the sequential binding of hGH, one model was used to characterize the bell-shaped relationships between hGH concentration and certain cell responses (14). Others have used this model, in conjunction with cell proliferation data, to estimate hGH site 1 and site 2 binding affinities and thus to make conclusions regarding the underlying structure-function relationships (15,16). Theoretically, the maximum response is always elicited at an hGH concentration equal to the site 1 dissociation constant (K D ) in this model, while the broadness of the peak is determined by the site 2 affinity and the degree to which intracellular effectors are limiting for signal transduction.…”

Growth hormone isoforms

“…However, the two isoforms are also equipotent in vitro in most assays (see above). The apparent discrepancy between full bioactivity despite low Site 1 binding affinity was postulated to be due to the strong interaction of two GHRs in their dimerization domain, thereby compensating for the weak Site 1 interaction [57]. The interaction of 20K-GH with the rat GHR is not known in the same detail; it may be significantly different from that with the human GHR.…”