Two Independent Histidines, One in Human Prolactin and One in Its Receptor, Are Critical for pH-dependent Receptor Recognition and Activation

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2010-12, Vol.285 (49), p.38524-38533
Main Authors: Kulkarni, Mandar V., Tettamanzi, M. Cristina, Murphy, James W., Keeler, Camille, Myszka, David G., Chayen, Naomi E., Lolis, Elias J., Hodsdon, Michael E.
Format: Article
Language:English
Subjects:
Cell Line, Tumor
Cytokine Action
Histidine - chemistry
Histidine - genetics
Histidine - metabolism
Human Prolactin
Human Prolactin Receptor
Humans
Hydrogen-Ion Concentration
JAK Kinase
Kinetics
Models, Molecular
Molecular Biophysics
pH Dependence
PKA
Prolactin - chemistry
Prolactin - genetics
Prolactin - metabolism
Protein Binding
Protein Structure, Quaternary
Receptors, Prolactin - chemistry
Receptors, Prolactin - genetics
Receptors, Prolactin - metabolism
STAT Transcription Factor
Surface Plasmon Resonance (SPR)
Thermodynamics
X-ray Crystallography
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M110.172072