Journal of Biological Chemistry
Volume 285, Issue 49, 3 December 2010, Pages 38524-38533
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Molecular Biophysics
Two Independent Histidines, One in Human Prolactin and One in Its Receptor, Are Critical for pH-dependent Receptor Recognition and Activation*

https://doi.org/10.1074/jbc.M110.172072Get rights and content
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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.

Cytokine Action
JAK Kinase
STAT Transcription Factor
Surface Plasmon Resonance (SPR)
X-ray Crystallography
Human Prolactin
Human Prolactin Receptor
pH Dependence
PKA

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*

This work was supported, in whole or in part, by National Institutes of Health Grant RO1 CA 108992 (to M. E. H.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–2, Figs. 1–5, and data.

1

Supported by National Institutes of Health Training Grant T32-HL007974-07.