The 20-Kilodalton (kDa) Human Growth Hormone (hGH) Differs from the 22-kDa hGH in the Complex Formation with Cell Surface hGH Receptor and hGH-Binding Protein Circulating in Human Plasma

Abstract

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.

Cooperative and differential effects of estrogen, prolactin, 22K and 20K human growth hormones on cyclin D1/PRAD1 gene expression in T‐47D human breast cancer cells

“…On the other hand, in addition to the major form of 22 kDa (22K hGH), human GH contains a variant form of 20 kDa (20K hGH) (10) derived from an alternatively spliced mRNA species (11). Recent reports have indicated that these two forms of hGH show different affinities to the GH and F'RL receptors and different bioactivities (12,13). In this study, we have analyzed the effects of human PRL (hPRL), 20K and 22K hGHs on the cyclin D1 gene expression in T-47D breast cancer cells, hPRL and 22K hGH Abbreviations used: GH, growth hormone; PRL, prolactin; 20K hGH, 20 kDa human growth hormone; 22K hGH, 22 kDa human growth hormone; hPRL, human prolactin; Ea, 17 13-estradiol; FBS, fetal bovine serum.…”

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

“…Because of the difficulty in preparing large enough amounts of highly purified 20K-hGH with authentic structure, its biological properties were unclear and controversial. Recently, we established an Escherichia coli secretion system for 20K-hGH with an authentic structure (6) and reported several properties of 20K-hGH (7)(8)(9)(10)(11).…”

“…Previous analyses showed that 20K-hGH formed an active 1:2 (hGH:hGHR) complex in a sequential manner as well as 22K-hGH, that is, the first hGHR binds to site 1 on hGH (this is designated "STEP1") and next the second one binds to site 2 (STEP2) (9,(11)(12)(13)(14). An active 1:2 (hGH:hGHR) complex formation is followed by tyrosine phosphorylation of hGHR and activation of intracellular signal transducer molecules (15)(16)(17).…”

“…Here we tentatively designate the stem region of hGHR, which is considered to be involved in receptor dimerization, "siteBP." Concerning the binding affinity between hGH and hGHR, we reported that KD1(22K) was smaller than KD1(20K) in the biosensor analysis (9) and that KDsite2(20K) was considered to be almost the same as KDsite2(22K), because the site 2 region of 20K-hGH was conformationally similar to 22K-hGH (11). On the other hand, the activity of 20K-hGH via hGHR is the same as that of 22K-hGH at low hGH concentration and even higher at high hGH concentration in cell proliferation assay (11).…”

“…Concerning the binding affinity between hGH and hGHR, we reported that KD1(22K) was smaller than KD1(20K) in the biosensor analysis (9) and that KDsite2(20K) was considered to be almost the same as KDsite2(22K), because the site 2 region of 20K-hGH was conformationally similar to 22K-hGH (11). On the other hand, the activity of 20K-hGH via hGHR is the same as that of 22K-hGH at low hGH concentration and even higher at high hGH concentration in cell proliferation assay (11). Therefore, we hypothesized that binding of 20K-hGH to the first hGHR could result in the transient 1:1 complex, which has a more favorable conformation for forming an active 1:2 complex especially at the siteBP region of hGHR.…”