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 hPRL⅐receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL⅐receptor 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 solventfilled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.Human prolactin (hPRL) is a 23-kDa protein hormone secreted both by the mammalian pituitary and numerous extrapituitary tissues. Based on similarity in quaternary structure and that of cognate receptors, prolactin is classified as a member of the larger family of hematopoietic cytokines, with highest homology to growth hormone (GH) and placental lactogen (1). Evidence suggests that hPRL functions as an autocrine-paracrine growth factor in cancers of the breast, prostate, and female reproductive tract. Both hPRL and its receptor (hPRLr) are expressed in a majority of breast cancers (2, 3), where their interaction affects various aspects of carcinogenesis (2-5). Cellular studies of hPRL signaling provide a molecular basis for the hPRL role in breast cancer proliferation (6 -10). Recent epidemiological studies have strengthened the link between elevated hPRL and breast cancer risk (10). Consequently, detailed molecular understanding of hPRL⅐receptor recognition and activation is critical to aid efforts to develop potential cancer intervention strategies.hPRL recognizes the hPRLr extracellular domain (ECD) and recruits two molecules of hPRLr via two distinct binding sites on the hPRL surface, i.e. site 1 (high affinity) followed by site 2 (low affinity) (11). Crystallographic structures of 1:1 (12) (hPRL antagonist⅐hPRLrECD) and 1:2 (13) (so called "affinity matured" hPRL⅐hPRLrECD) complexes reveal molecular details of the ϳ20% of the combined surface area buried within the high affinity binding interface. Cumulative work with mutagenic variants of hPRL further reduces the number of critical residue contacts for receptor binding (14 -16). Associated with formation of the heterotrimeric 1:2 (ligand⅐receptor) complex, induced conformational changes in the receptor intracellular domain brings two receptor-associated cytosolic tyrosine kinase molecules of Janus kinase-2 (JAK2)...

Phylogenetic and Functional Analysis of Histidine Residues Essential for pH-dependent Multimerization of von Willebrand Factor

“…The recycling mannose lectin ERGIC-53 binds cargo in the ER and releases it in the slightly more acidic ER-Golgi intermediate compartment (ERGIC), and cargo release requires protonation of conserved His 178 (12). A recent His-to-Ala mutagenesis study of the ligand-receptor binding interface showed that the evolutionarily conserved residues His 180 in human prolactin and His 188 in the prolactin receptor are critical for pH-dependent ligand binding at the cell surface and release in acidic endosomes (13). We have used phylogenetic comparisons to identify candidate pH-sensing His residues in VWF and homologous gel-forming mucins, which were functionally evaluated by mutagenesis.…”

A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling

“…In tumor contexts, the high glycolytic activity and proton extrusion by cancer cells is known to acidify the extracellular milieu (42). Knowing that PRL binding to the PRLR is strongly pH-dependent (43)(44)(45), the reduction of complex lifetime due to tumor acidification should reduce signaling to such an extent that it ultimately brings STAT5 activation into the regulatory range (Fig. 10).…”

Mutant Prolactin Receptor and Familial Hyperprolactinemia

“…1D). 11,14,18 His188 is found at the interface of the high-affinity binding site 1, 12,13,18 where it forms a polar contact (i.e., a noncovalent bond) with prolactin ( Fig. 1E) and contributes to a four-member histidine ring, formed by His188 in the prolactin receptor and three histidines of prolactin (His27, His30, and His180), which stabilizes ligand binding and determines the pH dependence of receptor activation.…”

“…13,18,19 Prolactin interacts with each extracellular domain of the dimeric prolactin receptor, first by means of a high-affinity (nanomolar range) binding site 1, and second, by means of a lowaffinity (micromolar range) binding site 2 ( Fig. 1D).…”

“…1E) and contributes to a four-member histidine ring, formed by His188 in the prolactin receptor and three histidines of prolactin (His27, His30, and His180), which stabilizes ligand binding and determines the pH dependence of receptor activation. 13 Structural modeling of the PRLR mutation associated with familial idiopathic hyperprolactinemia, which results in the His188Arg substitution, predicts the loss of both the polar contact with Asp183 in prolactin and the critical imidazole ring that contributes to the high-affinity binding site 1 (Fig. 1F).…”