Growth Hormone Binding Affinity for Its Receptor Surpasses the Requirements for Cellular Activity

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• 作者：Kenneth H. Pearce ; Jr. ; Brian C. Cunningham ; Germaine Fuh ; Tuula Teeri ; and James A. Wells
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：January 5, 1999
• 年：1999
• 卷：38
• 期：1
• 页码：81 - 89
• 全文大小：163K
• 年卷期：v.38,no.1(January 5, 1999)
• ISSN：1520-4995

文摘

The human growth hormone (hGH)-receptor interaction was used to study the relationshipbetween hormone-receptor affinity and bioactivity. hGH has two nonequivalent sites, called site 1 andsite 2, that bind two molecules of receptor in a sequential fashion. We produced both site 1 and site 2high-affinity hGH variants either by combining alanine mutants previously found to improve affinity atsite 1 or by random mutagenesis of residues in site 2 followed by phage display and receptor bindingselections. The two high-affinity variants, as well as one which combined them, were used in cellproliferation assays with FDC-P1 cells expressing the hGH receptor. Interestingly, none of these variantsproduced a change in the EC₅₀ for cell proliferation or the levels of JAK2 tyrosine kinase phosphorylation.Next we studied the effect of a reduction in site 1 affinity on cell proliferation. A systematic series ofhGH mutants were produced in which affinity for site 1 was reduced from 5- to 500-fold. Surprisingly,the EC₅₀ for cell proliferation was unaffected until affinity was reduced about 30-fold from wild-typehGH. Thus, native hGH-receptor affinity is much higher than it needs to be for maximal JAK2phosphorylation or cell proliferation. These studies begin to define basic functional tolerances for receptoractivation that need to be considered in the design of hGH mimics.