Growth hormone binding affinity for its receptor surpasses the requirements for cellular activity.

The human growth hormone (hGH)-receptor interaction was used to study the relationship between hormone-receptor affinity and bioactivity. hGH has two nonequivalent sites, called site 1 and site 2, that bind two molecules of receptor in a sequential fashion. We produced both site 1 and site 2 high-affinity hGH variants either by combining alanine mutants previously found to improve affinity at site 1 or by random mutagenesis of residues in site 2 followed by phage display and receptor binding selections. The two high-affinity variants, as well as one which combined them, were used in cell proliferation assays with FDC-P1 cells expressing the hGH receptor. Interestingly, none of these variants produced 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 of hGH 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-type hGH. Thus, native hGH-receptor affinity is much higher than it needs to be for maximal JAK2 phosphorylation or cell proliferation. These studies begin to define basic functional tolerances for receptor activation that need to be considered in the design of hGH mimics.