Date of Completion

4-18-2013

Embargo Period

4-18-2013

Keywords

prolactin, prolactin receptor, structural dynamics, receptor binding, metal binding, NMR, NMR relaxation, PRE, copper, zinc

Major Advisor

Jeffrey C. Hoch

Associate Advisor

Dmitry Korzhnev

Associate Advisor

Michael Gryk

Associate Advisor

Bing Hao

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

This thesis investigates the biophysical properties of the pituitary hormone prolactin. The prolactin receptor is overexpressed in many types of cancer tissues, making prolactin a molecule of pharmacological interest.

The nature of the interaction between prolactin and the prolactin receptor has been the subject of several studies, but results have been ambiguous. In an effort to clarify the lingering questions the formation of prolactin-prolactin receptor complexes in solution was studied using NMR and the wild type of prolactin and the extracellular domain of the prolactin receptor. It was shown that wild type prolactin is capable of forming both the binary and ternary complex with the extracellular domain of the prolactin receptor in solution, and it was determined which of the proposed binding affinities apply to wild type prolactin. It was demonstrated that the properties observed for the truncated and altered prolactin variants are shared by wild type prolactin. Lastly conformational entropy was ruled out as the driving force of the proposed allosteric effect.

Furthermore, metal binding by members of the growth hormone family has been suggested to play a role in facilitating reversible packing in secretory granules. Paramagnetic metal titration and chelation experiments on recombinant prolactin monitored by NMR relaxation were performed and unambiguously determined two metal binding sites on prolactin. The relative orientation of the two distinct metal binding sites, together with aggregation studies of hPRL mutants at biologically relevant pH values, enabled the proposal of a mechanism for metal-induced aggregation entailing intermolecular bridging between the metal binding sites.

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