Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation

Abstract

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

FIGURE 1.

SPR and thermodynamic modeling of WT and mutant hPRL to systematically assess the role of individual histidine residues in pH-dependent hPRLr binding. SPR performed with WT or mutant hPRL bound to a GLM sensor chip by standard amine coupling and purified recombinant hPRLrECD. Binding kinetics of the hPRL·hPRLrECD interactions obtained in quarter-unit increments in pH values over a range of pH 5.75–8.00 are shown. The affinity of H180A hPRL for hPRLrECD is ∼100-fold lower than that of hPRL at high pH, whereas a pH-dependent response is maintained. Best-fit simulation using observed binding affinity (by SPR) of WT or single site histidine mutants, H27A, H30A, and H180A, over a range of pH 5.5–8.0 are indicated by solid lines. The dashed line is an attempt to fit the WT data to a single pK_a model with the starting (i.e. high pH) fixed to the best-fit value from the two pK_a fit.

FIGURE 2.

Functional analysis of WT and single site histidine mutants of hPRL to evaluate their efficacy in pH-dependent hPRLr activation. T47D breast cancer cells, grown to ∼80% confluency in conditioned media, were treated with 1, 10, or 100 nm recombinant WT or mutant hPRL at the indicated pH for 10 min. Total protein cell lysates were used for immunoblotting to determine relative levels of total and phosphorylated STAT5, hPRLr, and α-tubulin. a, a saturable pH-dependent effect on STAT5 phosphorylation was observed over the pH range of 5.5–7.5. b, STAT5 phosphorylation by single site His mutants of hPRL (1 nm) at pH 7.5 indicates a critical role for His-180 in receptor activation. c, pH-dependent receptor activation by hPRL that lacks His-27, His-30, or His-180 (alone or in combination) is analogous to the binding affinity of each hPRL variant observed by SPR. Conc, concentration; Lig, ligand; rep, representative.

FIGURE 3.

Immunoblot analysis to determine the role of His-188 of hPRLr in pH-dependent receptor activation. CHO cells grown to ∼60% confluency were transfected with mammalian expression vectors for hPRLr (WT or H188A) as indicated. At ∼80% confluency (24-h post-transfection) cells were treated with WT or mutant hPRL (1 nm unless stated otherwise) at the indicated pH for 10 min. Total protein cell lysates were used for immunoblotting to determine relative levels of total and phosphorylated JAK2, total, and phosphorylated STAT5, hPRLr and α-tubulin. a, consistent pH-dependent JAK2 and STAT5, phosphorylation is observed only when the ligand·receptor combination retains His-180 of hPRL and His-188 of hPRLr. b, the pH-independent receptor activation (phosphorylated JAK2 and phosphorylated STAT5 levels) of the ligand·receptor pair lacking both His-180 of hPRL and His-188 of hPRLr is maintained at higher ligand concentration. Conc, concentration; Lig, ligand.

FIGURE 4.

Comparative analysis of the x-ray crystal structures of various mutant combinations of the hPRL antagonist·hPRLrECD complex. The upper left panel shows a schematic representation of the 1:1 WT antagonist (cyan)·WT hPRLrECD (magenta) complex with amino acid residues of the site 1 binding interface highlighted (gray). The upper right panel illustrates the hydrogen bonding network formed by the interactions of His-27, His-30, His-180 (hPRL), and His-188 (hPRLr). The bottom left panel shows superposition of the binding interface of WT·WT (gray) H27A·WT (magenta), and H30A·WT (cyan) complexes. The bottom right panel shows superposition of the binding interface of WT·WT (gray), H180A·WT (magenta), and WT·H188A (cyan) complexes.

FIGURE 5.

SPR and thermodynamic modeling of WT and H188A hPRLrECD to assess the role of His-188 in pH-dependent hPRL binding. SPR performed with WT or H180A hPRL bound to a GLM sensor chip by standard amine coupling and purified recombinant WT or H188A hPRLrECD. Binding kinetics of the hPRL·hPRLrECD interactions obtained in quarter unit increments in pH over a range of pH 5.75–8.00 are shown. Affinities of the H180A hPRL·WT hPRLrECD and WT hPRL·H188A hPRLrECD combinations are ∼100-fold lower than that of WT hPRL·WT hPRLrECD at high pH, whereas pH-dependent response is maintained. Loss of both His-180 and His-188 results in pH-independent receptor binding along with loss in affinity. Solid lines indicate best-fit simulation using observed binding affinity (by SPR) of WT hPRL or H180A hPRL for WT hPRLrECD or H188A hPRLrECD over a range of pH 5.5–8.0. The asterisk indicates data from Fig. 1, included to ensure reproducibility and consistent comparison.