The kinetics of binding human prolactin, but not growth hormone, to the prolactin receptor vary over a physiologic pH range

A member of the family of hematopoietic cytokines, human prolactin (hPRL) serves a dual role both as an endocrine hormone and as an autocrine/paracrine cytokine or growth factor. During investigation of the solution structural properties of hPRL, we have noted a surprising pH dependence of its structural stability over a range from approximately pH 6.0 to pH 8.0. An analysis of backbone atom NMR chemical shift changes and backbone amide hydrogen-deuterium exchange rates due to titration of the solution pH over this same range, along with calculations of protein surface electrostatic potential, suggests the possible involvement of a localized cluster of three His residues (27, 30, and 180), which comprise a portion of the high-affinity receptor-binding epitope. Surface plasmon resonance analysis of the interaction between hPRL and the extracellular domain (ECD) of the hPRL receptor reveals a selective 500-fold change in the dissociation rate between pH 8.3 and pH 5.8. In comparison, the interaction of hGH with the same receptor ECD did not demonstrate any significant dependence on pH. We also present an initial investigation of the pH dependence of hPRL function in rat Nb2 cell proliferation assays and a STAT5 luciferase gene reporter assay in the T47D human breast cancer cell line, whose results are consistent with our biophysical studies. The potential implications of this variation in hPRL's structural stability and receptor-binding kinetics over this physiologic range of pH are discussed.