The hydronium ion (H₃O⁺) concentration, most often expressed as its negative log (i.e. pH), or more simply as the solution acidity, represents a fundamental property of any biologic environment. Within the human body, the acidities of compartmentalized fluids are tightly regulated and generally vary over a range from pH 5 – 8, corresponding to hydronium ion concentrations between 10^-5 and 10^-8 M. As such, we note its similarity to other physiologic ions, whose localized concentrations fluctuate over equivalent ranges and regulate specific biologic activities, e.g. the role of the Na⁺ ion in neuronal axon depolarization or the Ca²⁺ ion and muscle cell contraction, amongst many others. In nearly all of these cases, the fluctuating ion binds to specific protein sites, with dissociation constants in the same 10^-5 to 10^-8 M range, to allosterically alter protein functions. The molecular basis and cellular importance of these allosteric ion-binding sites have often been well characterized, such as the structure and function of the Ca²⁺-sensing “EF hand” domain. In contrast, molecular characterization of the hydronium ion’s interactions with proteins is less well defined, as it is much more ubiquitous, with many titratable groups (i.e. the ionizable amino acids) found on each and every protein in the human genome. Nevertheless, during evolution proteins must have become similarly “tuned” to functionally respond to regulated variations in local pH, a hypothesis that has recently been supported by genome-wide analyses. As pathology is frequently associated with alteration in the pH of the local environment, experimental exploration of the biophysical basis of pH-dependent regulation of protein function and its cellular implications will improve our understanding of both basic human biology and associated pathologic processes.

Figure 1: Approximate variation in pH during the functional lifecycle of secreted protein hormones.	Figure 2. Kinetics for binding of both hPRL and hGH to the hPRLr-ECD. SPR results are plotted as the dissociation and association rate constants for each protein as a function of pH. Equilibrium binding constants are illustrated by dotted iso-energetic contour lines.

We have chosen to focus on two secreted protein hormones to investigate the biophysical and functional consequences of allosteric proton-binding (i.e. pH-dependence), as the biology of secreted proteins is evidently regulated by pH (see Figure 1). Additionally, our laboratory previously discovered a dramatic dependence on pH to their structural and functional properties: the structural stability of hPRL and its affinity for the extracellular domain (ECD) of its receptor (hPRLr) vary over the relatively narrow range from pH 6 – 8. Figure 2 displays the results of surface plasmon resonance (SPR) experiments on the binding of both hPRL and hGH to the hPRLr-ECD. Upon acidification from pH 8 to 6, the receptor-binding affinity of hPRL weakens by nearly one thousand-fold; whereas, binding of hGH to the same hPRLr-ECD remains nearly unchanged over this entire pH range. Because the latter interaction required the presence of 50 µM Zn²⁺, we subsequently repeated the hPRL-binding studies in the presence of Zn²⁺ and found no change in the results; thus, the significant difference in pH-dependence between hPRL and hGH-binding to the hPRLr-ECD relates directly to a structural difference in the two hormones.

Basic Biology of hPRL and hGH. Along with the less well characterized placental lactogen, PRL and GH constitute a trio of homologous protein hormones, which are both functionally and evolutionarily related. These three proteins are part of the family of hematopoietic cytokines, which includes erythropoietin, granulocyte-colony stimulating factor, interleukin-6, and others. Proteins in this family share a common structural fold (the up-up-down-down four α-helical bundle) and recognize a conserved family of cell surface receptors. Functionally, mammalian PRL and GH have been characterized as both endocrine hormones and autocrine-paracrine growth factors/cytokines. As endocrine hormones, they are expressed by the anterior pituitary under control of the “Pit-1” promoter and packaged into dense cores of secretory granules, whose release is regulated by the hypothalamus. After diffusion into the bloodstream, GH acts distally to regulate growth and anabolic function, while endocrine PRL regulates reproductive function and lactation. However, both PRL and GH are also synthesized in a variety of extrapituitary tissues including kidney, spleen, breast, prostate, placenta, uterus, endothelium, immune cells and the central nervous system. Extrapituitary hormone expression utilizes an alternative transcriptional promoter and splicing pathway; secretion of the hormones into interstitial fluid is constitutive, where they act locally to regulate cellular growth and differentiation. These distinct biological differences suggest an alternative physiological role for extrapituitary PRL and GH. Additionally, a variety of post-translationally modifications have also been identified, some of which have dramatically different activities than the unmodified hormone. We have hypothesized for the susceptibility of hPRL and hGH to enzymatic modification to be regulated by pH-dependent effects on localized conformation and structural stability.

Role of hPRL function in human cancer. Evidence suggests PRL functions as an autocrine-paracrine growth factor in cancers of the breast, prostate and female reproductive tract. Hyperprolactemia has been associated with increased mammary tumorigenesis in rodents and administered PRL increases the size and incidence of spontaneous and induced tumors. Both PRL and its receptor are expressed in a majority of human breast cancers, where it has a mitogenic effect, induces angiogenesis, and increases cancer cell motility. A majority of clinical breast tumors examined express the PRLr and malignant breast tissues over-express PRLr compared to contiguous normal tissue. Cellular studies of PRL signaling have provided a molecular basis for PRL’s role in breast cancer proliferation via crosstalk with the estrogen receptor signaling and the EGFr family’s signaling pathways. Multiple epidemiologic studies have strengthened the link between PRL and breast cancer risk and a recent similar association with gynecologic cancers has been suggested. Lastly, the overall effect of hPRL signaling appears to counter the effects of anti-cancer therapeutics, most likely attributable to its function as a stimulator of cell proliferation and inhibition of apoptosis, suggesting that the development of an effective antagonist or suppressor of hPRL function will enhance the efficacy of existing chemotherapeutic regimens.

Figure 3: Structural depictions of hPRL and hGH complexed with their receptors. Starting in the upper left, a backbone ribbon diagram of the trimeric complex of hGH and two identical copies of the hGHr-ECD (PDB: 1HWG) is shown, with the hPRL and hGH receptor-binding specifities diagrammed underneath. The upper right panel displays the locations of nine histidines in the tertiary structure of hPRL (PDB:2Q98). Lastly, the bottom two panels present the intermolecular interfaces for the hPRLr-ECD bound to the high affinity site of hGH (left) and hPRL (right), focusing on the predominant interactions of histidines (see text).

Recognition and activation of cell surface receptors. Both PRL and GH recognize and activate the prolactin receptor (PRLr) and growth hormone receptor (GHr). Both are characterized by a single transmembrane-spanning region dividing the extracellular ligand-binding domain (ECD) from the cytoplasmic signaling domain. Activation requires the formation of a trimeric complex between two identical receptor molecules and a single protein hormone involving a high affinity interaction (1 – 10 nM) between one receptor ECD and “site 1” on the protein hormone, along with a second, lower affinity (1 – 50 µM) binding event (site 2) on the opposite face of the hormone (see Figure 3). The large difference in binding affinity between the two sites results in a self-inhibitory phenomenon (similar to the immunologic ‘hook effect’) at sufficiently high hormone concentrations where all the available receptor molecules become sequestered in 1:1 hormone:receptor complexes. The intermolecular interface between both pairs of hormone-receptor complexes involves a cluster of conserved histidines (H27, H30 and H180 in hPRL), which are a major focus of our work. Despite conservation of these histidines from the binding-interface in the hGH-hPRLr complex, the pH dependence of the interaction is dramatically different. For the human orthologues, hPRL is highly specific for recognition of the hPRLr; whereas, hGH can activate both receptors; however, recognition by hGH requires the presence of 10 – 100 µM Zn²⁺. The physiologic relevance of hGH-binding to the hPRLr is uncertain. Although plasma Zn²⁺ levels do reach as high as 15 µM, syndromes of GH excess in humans (i.e. acromegaly) are not associated with clinical signs of hyperprolactinemia.

We desire a fundamental description of the molecular mechanism for the pH-dependence of hPRL and hGH receptor recognition, with the eventual goal of identifying mutagenic variants with altered pH dependence both as a validation of our biophysical model and as an experimental tool to be used in cellular studies. Our approach will be to first experimentally define the site-specific protonation reactions for both the free and complexed proteins. The derived protonation constants (i.e. pKa values) will subsequently be utilized to construct a thermodynamic model for the pH dependence of receptor-binding affinity. We will also determine high resolution tertiary structures of the complexed proteins and computationally model the electrostatic interactions responsible for the perturbations in site-specific protonation that give rise to the pH dependence of receptor recognition. The insight gained from this analysis will be used to iteratively design and experimentally validate mutagenic variants with altered pH dependence.