Differential effects of zinc on functionally distinct human growth hormone mutations.

Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 microM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose-response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose-response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose-response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.

Human growth hormone site 2 lactogenic activity requires a distant tyrosine164.

Comparison of crystallographic structures of human growth hormone, either bound to the prolactin receptor or free of receptors, reveals that human growth hormone binding to the prolactin receptor at site 1 is associated with a structural change in human growth hormone that influences the organization of residues which constitute site 2. We have identified Tyr164 as a residue that is critical for the propagation of this structural rearrangement. Tyr164 is a structural epitope for site 1 and is distal to site 2. Mutation of Tyr164 to glutamic acid (Y164E) does not affect the somatotrophic activity, absorption or fluorescence spectra or binding to the human prolactin receptor when compared to wild-type human growth hormone, indicating the subtle effects of the mutation. Lactogenic assays using extended concentrations of Y164E human growth hormone produce dose-response curves that are characterized by a right-shifted agonist phase and an unchanged antagonist phase when compared to wild-type human growth hormone. These results indicate that Tyr164 is required for the lactogenic activity of human growth hormone and that mutation to glutamic acid disrupts the lactogenic function of site 2.