Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization.
Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.
Institute for Molecular Bioscience and School of Biomedical Sciences, University of Queensland, St. Lucia 4072, Australia.
Yu
Y
Wan
Andrew
A
McDevitt
Bojiang
B
Shen
Mark L
ML
Smythe
Michael J
MJ
Waters
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D049109:Cell Proliferation; D018360:Crystallography, X-Ray; D004058:Diffusion; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D013006:Growth Hormone / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013552:Swine; D014364:Tryptophan / Q000737:chemistry
D011994:Recombinant Proteins; D014364:Tryptophan; D013006:Growth Hormone; D008239:Lysine; D010100:Oxygen