One of the key techniques used for deciphering the mechanism of action of hGH was a high-resolution analysis of the binding event by alanine-scanning mutagenesis (13,24,25). Initially, single mutations were made at all side-chains (62 in total) between residues 2-19, 54-74, and 167-191. Binding analysis of each of these hGH mutants showed that only about 12 residues gave a fourfold lower binding affinity for the hGHR compared to wild-type hGH. When a similar set of mutants was tested for the ability to dimerize the hGHR using a fluorescence-quenching assay, one subset of mutations affected binding of the 1:1 complex (Site 1), whereas a different set of mutations had no effect on 1:1 binding, but did have an effect on receptor dimerization (Site 2).
To further investigate the role of individual residues in the 1:1 hGH-hGHR binding event, equilibrium and kinetic data were obtained using surface plasmon resonance on a Pharmacia BIAcore device (25). To study Site 1 exclusively, a receptor mutant (S237C) was coupled to the sensor chip in a manner that prevents dimerization on hGH
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