How and Why Does pH Regulate Cytokine-Receptor Recognition?
The Hydronium Ion
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.
We note its similarity to other physiologic ions, whose localized concentrations fluctuate over equivalent ranges and regulate specific biologic activities. In general, 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 allosteric ion-binding sites have often been well characterized: the Ca2+-binding “EF hand" domains, Zn2+-finger proteins, etc. 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 encoded by the human genome.
Nevertheless, we believe it is evident that during evolution proteins have become “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.
Functional lifecycles of secreted protein hormones.
Secreted polypeptide hormones experience multiple regulated changes in local pH during their complex functional lifecycles. Following initial ribosomal synthesis and insertion into the endoplasmic reticulum (ER), proteins targeted for secretion are initially exposed to acidity near intracellular pH ~ 7.3. During their subsequent journey through the Golgi apparatus, the pH is actively lowered to a range of 5.5 – 6.0 and, at the final trans-Golgi, the proteins may either be trafficked directly to the cell membrane or packaged into secretory granules, reserved for later secretion. Research has demonstrated a primary role for a final acidification of the trans-Golgi to around pH 5.0 in triggering the spontaneous aggregation of protein hormones into the dense granules required for packaging into secretory vesicles. Upon secretion into extracellular fluid, protein hormones experience an immediate change in solution pH, approaching neutrality in healthy tissues, where aggregated proteins derived from secretory granules nearly instantaneously dissolve. Depending on their functional role, secreted hormones may act locally or diffuse into the bloodstream for systemic distribution. Ultimately, their action at target cells will be regulated by the local extracellular microenvironment. Although the pH of circulating blood is tightly controlled over a narrow range (7.35 – 7.45), the acidity of the extravascular space (interstitital fluid) varies more widely. Careful experimental measurement of interstitial pH in healthy tissue reveals a range of acidity from pH 7.1 – 7.7. Additionally, the extracellular pH of pathologic tissue has been consistently found to be more acidic than surrounding stroma, by 0.5 – 1.0 pH units. After binding to their target cell surface receptors, complexed ligand-receptor complexes are rapidly endocytosed. During step-wise maturation of the resulting endosome, the intraendosomal pH is actively acidified until a final value near pH 5.5 in the so-called ‘late endosome’ or ‘multi-vesciular body’. Activation of intracellular signaling pathways continues from the cytoplasmic side of early endosomes, whose pH remains near neutral. Evidence has accumulated demonstrating a complex intertwining between the endocytosis and intracellular signaling pathways. The overall functional effect of ligand-mediated activation of a cell surface receptor depends very much on the molecular interactions and structural modifications occurring after internalization of the complexed ligand-receptor complexes and throughout the subsequent endocytic pathway, terminating in either lysosomal degradation or recycling of the proteins back to the extracellular environment. The pH of the endocytic compartment is under active control by the cell and we hypothesize acts as a critical regulator of endocytic trafficking.
Human Prolactin (hPRL)and Growth Hormone (hGH)
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, above). Our laboratory previously discovered a dramatic dependence on pH to the structural and functional properties of these two proteins. The results of surface plasmon resonance (SPR) experiments on the binding of both hPRL and hGH to the hPRLr-ECD are displayed in the figure to the left. 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 essentially 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.
The structural stability of hPRL is similarly dependent on pH, with greater stability in more alkaline solution.
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Adaptations of proteins to cellular and subcellular pH. Garcia-Moreno B. J Biol. 2009;8(11):98. Epub 2009 Dec 2. Review.PMID: 20017887. Free Full Text