The binding between the stem regions of human growth hormone (GH) receptorcompensates for the weaker site 1 binding of 20-kDa human GH (hGH) than that of 22-kDa hGH

Despite the lower site 1 affinity of the 20-kDa human growh hormone (20K-hG H) 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 acti vity at high hGH concentration. This study was performed to elucidate the r eason 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 o f 22K-hGH in the sequential binding with hGHR, To demonstrate it, we prepar ed 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 compa red 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 thre e 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 mathem atical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attribut ed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGH R between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lowe r than that of 22K-hGH.