Abstract
Excess circulating human growth hormone (hGH) in vivo is linked to metabolic and growth disorders such as cancer, diabetes, and acromegaly. Consequently, there is considerable interest in developing antagonists of hGH action. Here, we present the design, synthesis, and characterization of a 16-residue peptide (site 1-binding helix [S1H]) that inhibits hGH-mediated STAT5 phosphorylation in cultured cells. S1H was designed as a direct sequence mimetic of the site 1 mini-helix (residues 36–51) of wild-type hGH and acts by inhibiting the interaction of hGH with the human growth hormone receptor (hGHR). In vitro studies indicated that S1H is stable in human serum and can adopt an α-helix in solution. Our results also show that S1H mitigates phosphorylation of STAT5 in cells co-treated with hGH, reducing intracellular STAT5 phosphorylation levels to those observed in untreated controls. Furthermore, S1H was found to attenuate the activity of the hGHR and the human prolactin receptor, suggesting that this peptide acts as an antagonist of both lactogenic and somatotrophic hGH actions. Finally, we used alanine scanning to determine how discrete amino acids within the S1H sequence contribute to its structural organization and biological activity. We observed a strong correlation between helical propensity and inhibitory effect, indicating that S1H-mediated antagonism of the hGHR is largely dependent on the ability for S1H to adopt an α-helix. Taken together, these results show that S1H not only acts as a novel peptide-based antagonist of the hGHR but can also be applied as a chemical tool to study the molecular nature of hGH–hGHR interactions.
Highlights
Pituitary gland, human growth hormone (hGH) can be released peripherally by immune, neural, reproductive, alimentary, and respiratory tissues and in the integumentary, muscular, skeletal, and cardiovascular systems [5,6,7,8,9]
To reduce the influence of induced or additive binding effects resulting from the preassociation of native hGH to the human growth hormone receptor (hGHR), we focused our attention on designing a peptide-based antagonist that would target the incipient interaction
Targeting this site for inhibition was deemed suboptimal because the inhibitor peptide would have to compete sterically with amino acid residues of the hGHR that are involved in receptor dimerization
Summary
Pituitary gland, hGH can be released peripherally by immune, neural, reproductive, alimentary, and respiratory tissues and in the integumentary, muscular, skeletal, and cardiovascular systems [5,6,7,8,9]. We reasoned that site 1-binding interactions between hGH and hGHR could be inhibited using a peptide mimetic that was based on the mini-helix (residues 38–47) found in the “large loop” of hGH. To reduce the influence of induced or additive binding effects resulting from the preassociation of native hGH to the hGHR, we focused our attention on designing a peptide-based antagonist that would target the incipient (site 1) interaction.
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