Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor

Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor

Bibliographic Details
Main Author: Liu, Wei
Language:English
Published: The Ohio State University / OhioLINK 2012
Subjects:
Biochemistry
prolactin receptor
human
dimerization
disulfide
structure function
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1323893195
id ndltd-OhioLink-oai-etd.ohiolink.edu-osu1323893195
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Biochemistry
prolactin receptor
human
dimerization
disulfide
structure function
spellingShingle Biochemistry
prolactin receptor
human
dimerization
disulfide
structure function
Liu, Wei
Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
author Liu, Wei
author_facet Liu, Wei
author_sort Liu, Wei
title Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
title_short Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
title_full Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
title_fullStr Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
title_full_unstemmed Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
title_sort structural basis and functional impact of ligand-independent dimerization for human prolactin receptor
publisher The Ohio State University / OhioLINK
publishDate 2012
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1323893195
work_keys_str_mv AT liuwei structuralbasisandfunctionalimpactofligandindependentdimerizationforhumanprolactinreceptor
_version_ 1719430386991235072
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu13238931952021-08-03T06:04:18Z Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor Liu, Wei Biochemistry prolactin receptor human dimerization disulfide structure function <p>The human prolactin receptor (hPRLr) is a member of the class 1 cytokine receptor family, which also includes growth hormone receptor (GHr) and erythropoietin receptor (EPOr). The classic mechanism for class 1 cytokine receptor activation describes that ligand-induced receptor dimerization triggers downstream signaling and is supported by biochemical and biophysical evidence. However, the recent discoveries of ligand-independent dimerization of class 1 cytokine receptors in plasma membrane, including hPRLr, hGHr, and mouse EPOr, have challenged this classic mechanism. Several mechanistic models have been proposed for the activation of GHr and EPOr, including the scissor model, the piston model, and the rotation model. In contrast, it is unknown if hPRLr shares a similar mechanism. The specific amino acid residues that mediate ligand-independent hPRLr dimerization have not been determined, although the transmembrane (TM) domain has been suggested to be important. The role of ligand-independent hPRLr dimers in hPRLr activation is unclear. Furthermore, the presence of ligand-independent hPRLr dimers does not rule out the classic mechanism, because hPRLr may exist in equilibrium between monomers and preformed dimers in plasma membrane. This dissertation has focused upon the structural basis and the functional impact of ligand-independent dimerization of hPRLr.</p><p> We aimed to systematically evaluate in hPRLr the proposed models for class 1 cytokine receptor activation. An extensive series of alanine or glycine insertions were introduced at the junctions between the TM domain and either the extracellular or intracellular domain to manipulate the relative orientations of different hPRLr domains. The basal and ligand-stimulated activities of these hPRLr insertion variants were examined in transiently transfected 293T cells. Our data demonstrate that altering the spatial relationships of hPRLr domains does not induce constitutive activity or impair ligand-induced activation. Such results do not support the rotation or piston model for hPRLr.</p><p> We also identified a population of covalently linked ligand-independent hPRLr dimers that are redox-sensitive and investigated the involvement of intermolecular disulfides in ligand-independent hPRLr dimerization. Twelve cysteines in various domains of hPRLr were replaced with serines, and the dimerization status of these hPRLr mutants was examined under reducing and non-reducing conditions. Iodoacetamide, an alkylation reagent for cysteine, was employed to distinguish in vivo disulfides from ex vivo disulfides. Our data indicate that multiple cysteines from different domains of hPRLr, including but not limited to C184, C225, and C242, participate in forming intermolecular disulfides in ligand-independent hPRLr dimerization.</p><p> We next examined the role of these disulfide-linked hPRLr dimers in hPRLr activation. Abolishing the formation of ligand-independent disulfide-linked hPRLr dimers by removing twelve C-terminal cysteines did not impair ligand-induced activation or affect the speed of activation, indicating that disulfide-linked hPRLr dimers are not required for hPRLr activation. Furthermore, wild-type disulfide-linked hPRLr dimers were not phosphorylated after ligand stimulation. Only hPRLrs that were not covalently linked became phosphorylated. We conclude that disulfide-linked hPRLr dimers are unlikely to participate in hPRLr activation. Collectively, the data in this dissertation suggest the validity of the classic mechanism of ligand-induced dimerization for hPRLr activation and illustrate the uniqueness of hPRLr in this receptor family.</p> 2012-01-05 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1323893195 http://rave.ohiolink.edu/etdc/view?acc_num=osu1323893195 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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