In spite of recent advance in understanding of the stoichiometry of 22-kDa human GH (22K-hGH) with cell surface hGH receptor (hGHR) and hGH-binding protein (hGH-BP) circulating in human plasma, that of 20-kDa hGH (20K-hGH) is poorly understood. To clarify this, mouse pro-B Ba/F3 cells stably expressing the full-length hGHR (Ba/F3-hGHR) and both recombinant and native hGH-BP were used in this study. Cell proliferation assay revealed that the two hGH isoforms increased Ba/F3-hGHR cells to the same extent in a dose-dependent manner at 0.1 pM-10 nM. However, the self-inhibition observed in 20K-hGH at 5 microM was significantly less than that in 22K-hGH. Furthermore, addition of 1 and 10 nM recombinant hGH-BP caused a slight inhibition in 20K-hGH, but a drastic inhibition in 22K-hGH. Gel filtration chromatography of mixtures of 20K-hGH with recombinant hGH-BP clearly demonstrated that 20K-hGH formed a 1:2 (hGH:hGH-BP) complex efficiently but no detectable 1:1 complex in any conditions. Supporting data were also obtained with native hGH-BP. Computer-aided homology modeling of 20K-hGH has provided speculative data that the conformational change caused by deletion of 15 residues may occur only in the loop between helix 1 and helix 2, resulting in the reduction of its site 1 affinity. In conclusion, 20K-hGH possesses a unique property for forming a 1:2 complex to the same extent as 22K-hGH but has difficulty in forming a 1:1 complex, which might be attributed to the conformational change restricted to its site 1 region.

Previously we have demonstrated that 20-kDa human GH (20K-hGH) is a full agonist for hGH receptor (hGHR) even though its complex formation with hGHR and hGH-binding protein differs from that of 22-kDa human GH (22K-hGH). In this study, we focused on the effect of 20K-hGH on human PRL receptor (hPRLR). To elucidate the effects of 20K-hGH on hPRLR and compare them with those of 22K-hGH, we prepared two cells stably expressing full-length hPRLR, Ba/F3-hPRLR and CHO-hPRLR. In the proliferation of Ba/F3-hPRLR cells, which can grow in a dose-response to lactogenic hormones, both 20K- and 22K-hGH exhibited bell-shaped curves in the absence of exogenous zinc ion (Zn2+); however, the curve of 20K-hGH was shifted to a 10-fold higher concentration than that of 22K-hGH in view of EC50 value (the EC50 of 20K- and 22K-hGH were 15 nM and 1.5 nM, respectively). Addition of Zn2+ up to 25 microM increased the activities of both 20K- and 22K-hGH; however, the enhancement by Zn2+ was greater in 20K-hGH than in 22K-hGH, thereby the activities of both hGH isoforms reached the same level at 25 microM Zn2+. Nevertheless, in the presence of 0.25-1 microM free Zn2+, which is equal in human serum, the activity of 20K-hGH was still lower than that of 22K-hGH. The modest effect of 20K-hGH on activating hPRLR in the absence of Zn2+ was confirmed in the rat serine protease inhibitor 2.1 (Spi2.1) gene promoter activation and JAK2/Stat5 tyrosine phosphorylation in CHO-hPRLR. In addition, in human breast cancer cell T-47D, 20K-hGH was proved to stimulate Stat5 tyrosine phosphorylation to much lower degree than 22K-hGH via not hGHR but hPRLR. Taken together, our data suggest that 20K-hGH may be a weaker agonist for hPRLR than 22K-hGH in the human body; therefore 20K-hGH may alleviate the hPRLR-mediated side-effects such as breast cancer when administered to human body.

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.

Even though 20K human growth hormone (20K-hGH) has 3–10% binding affinity for the rat liver and adipose tissue microsomes as compared to 22K-hGH, it was also reported that 20K-hGH has the same potency as 22K-hGH in the hypophysectomized rat weight gain assay. In order to investigate the reason why such controversial data exist, we have studied 20K- and 22K-hGH using the rat GH receptor extracellular domain (rGHR-ECD) and full-length rGHR. When we examined the complex formation of rGHR-ECD with 20K- and 22K-hGH in gel filtration assay, 20K-hGH formed no complex while 22K-hGH formed a 1:1 complex. Next, rGHR cDNA was introduced into Ba/F3 cells and CHO-K1 cells, and stable transfectants (Ba/F3-rGHR and CHO-rGHR) were established. In the proliferation of Ba/F3-rGHR cells, 20K-hGH had 10-fold lower activity than 22K-hGH, which is consistent with their affinities for rGHR. But surprisingly, in the Spi2.1 gene promoter activation in CHO-rGHR cells, 20K- and 22K-hGH had the same activity, which was found not only in stable CHO-rGHR clones but also in CHO-K1 cells transiently expressing rGHR. In conclusion, these results indicate that cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rGHR.