The 20-Kilodalton (kDa) Human Growth Hormone (hGH) Differs from the 22-kDa hGH in the Effect on the Human Prolactin Receptor

Abstract

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.

Structure and function of a new class of human prolactin antagonists

“…The structure of 20 kDa hGH is not known, but based on the structures of 22 kDa hGH [36,37], the loss of the 15 residues reduces the length of the backbone connecting helices 1 and 4, constraining the possible spatial relationships between these two helices. Tsunekawa et al have shown that although 22 kDa hGH has both somatotrophic and lactogenic activities; 20 kDa hGH retains only somatotrophic activity [38]. We have confirmed the requirement for residues 32 through 46 for hGH lactogenic activity and expanded this work into hPRL [39].…”

“…Based on the work of Tsunekawa et al [38] in the 20 kDa form of hGH, we prepared a similar deletion mutant in hPRL (Δ41-52 hPRL) [39] and were surprised to observe that this deletion provided a substantially greater reduction of lactogenic activity than that provided by the 20 kDa hGH. We also realized that this deletion contained some of the residues that we had identified as being critical for activity and necessary to functionally couple site 1 with site 2 in hPRL.…”

Cellular Activities of 20K- and 22K-hGH Do Not Necessarily Correlate with Their Binding Affinities for Rat GH Receptor

“…The data observed in this study holds true in rat GHR, however it should be noted that cell proliferation and the gene activation potencies of 20K- and 22K-hGH well correlated each other in human GHR [12, 14]and also human prolactin receptor [29]. It remains unclear what causes the unrelated cellular activities especially in rat GHR, however the different signaling pathways may be relevant to it.…”

The 20-kD Human Growth Hormone Reduces Body Fat by Increasing Lipolysis and Decreasing Lipoprotein Lipase Activity

“…Uchida et al [4]have previously developed an efficient 20K-hGH production system and have confirmed that the resultant product has a high purity and the expected amino acid sequence, which is based upon the splice site for the larger hGH. This recombinant authentic 20K-hGH is a strong agonist for growth promotion in hypophysectomized rats [4], but in comparison with 22K-hGH it is a defective agonist for actions mediated by the human prolactin receptor [5]. 20K-hGH acts in a similar manner as the full-length human growth hormone receptor (hGHR) agonist, forming an active 1:2 20K-hGH:hGHR complex with little formation of the inactive 1:1 complex [6, 7].…”