Molecular/structural basis of ligand-induced selective signalling of G protein-coupled receptors in reproduction/cancers

Lead Research Organisation: MRC Human Reproductive Sciences Unit

Abstract

Neuropeptide hormones play important roles in regulation of the mammalian reproductive system. In the pituitary gland, GnRH stimulates release of gonadotropins which in turn regulate sex steroid hormones and production of eggs or sperm. Interestingly, GnRH has a direct inhibitory effect on the growth of cancer cells in reproductive tissues. As a result of its pivotal role, GnRH analogues are widely employed in the treatment of infertility and hormone-dependent diseases. ||GnRH analogues exert their pharmacological effects through binding to a G protein-coupled receptor on cell surfaces. This binding triggers a change of the receptor 3D-structure enabling it to recognise and activate G proteins and their downstream signalling inside of cells and resulting in changes in function, shape or mobility of cells.||We use molecular modelling and site-directed mutagenesis studies to reveal how GnRH analogues bind to the human GnRH receptor and how GnRH binding triggers a change of the receptor 3D-structure and how this change recognises and activates different G proteins and their intracellular signalling. This study will enhance our understanding of the receptor activation mechanisms and identify novel signalling pathways and assist development of novel GnRH analogues with improved pharmacologic specificity and reduced side-effects caused by off-target cross-activity.

Technical Summary

The gonadotropin-releasing hormone (GnRH) receptor is central to the control of the entire reproductive system and therefore a critical target for therapeutic intervention. Our recent discovery that different GnRH analogues have differential effects on intracellular signalling pathways has major implications. The first is that the human GnRH receptor can assume a number of different active conformations that mediate different signalling pathways. The second is that GnRH ligand binding breaks intramolecular constraint networks and creates new sets of inter- and intra-molecular contacts that stabilise the receptor in different active conformations mediating different signalling. The third is that different GnRH analogues make a number of common and ligand-specific inter-molecular contacts with the receptor that stabilise distinct receptor active conformations which give rise to differential signalling.||Due to the difficulty of crystallising GPCRs, our understanding of ligand-induced receptor conformational changes associated with receptor activation relies on biophysical and biochemical studies. Using molecular modelling, receptor mutagenesis and a range of substituted GnRH analogues, we have made progress in identifying the inter- and intra-molecular contact residues involved in GnRH ligand binding and receptor activation. In this programme, we will use molecular modelling, sequence analysis of co-ordinated evolutionary changes of amino acids among GnRH receptors and naturally occurring hGnRH receptor mutations to identify potential inter- and intra-molecular contact residues. This information will be used for further studies using site-directed mutagenesis and GnRH analogues with single amino acid substitution to define their functions in the receptor conformational switch and consequent signal selectivity.||The GnRH receptor has been proposed to couple with different G proteins to mediate different physiological and pharmacological effects. We are endeavouring to clarify these interactions at molecular and atomic level, and to unravel novel GnRH receptor signalling pathways and the mechanisms of GnRH analogue-stimulated antiproliferative effects on cancer cells. These studies will enhance our understanding of GnRH receptor activation mechanisms and the efficacy and side effects of GnRH analogues currently employed in a diversity of clinical treatments and assist in the development of novel therapeutic signal-selective GnRH analogues which act via receptor conformational selection.||We are also extending our research to other GPCRs such as Kisspeptin/GPR54, GnIH/GPR147 and Estrogen/GPR30 which also play important roles in reproductive health and illness.

Publications

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Millar RP (2008) Diversity of actions of GnRHs mediated by ligand-induced selective signaling. in Frontiers in neuroendocrinology

 
Title GnRH receptor models 
Description We have built models of the human GnRH receptor in inactive and active conformational states which were refined using extensive site-directed mutagensis results obtained in our own and other labs. 
Type Of Material Data analysis technique 
Year Produced 2007 
Provided To Others? Yes  
Impact About 50% of clinical drugs target G protein-coupled recpetors (GPCRs), but only a few 3-dimensional structures of GPCRs have been available. We aim to build reliabe GnRH receptor models which could be used for in-silico screening of ligands. If success, this can be extended to other GPCRs. 
 
Title Molecular Models of Kisspeptin and GPR54 
Description Molecular modelling 
Type Of Material Data analysis technique 
Year Produced 2009 
Provided To Others? Yes  
Impact to predict the potential interactions of kisspeptin with its receptor which can be tested by mutagenesis studies and assist in development of novel kisspeptin analogues. 
 
Description A. Kemal Topalogu 
Organisation Cukurova University
Department Division of Paediatric Endocrinology and Metabolism
Country Turkey 
Sector Academic/University 
PI Contribution Characterization of the mutant receptor in vitro.
Collaborator Contribution Identified a natural mutation of the GnRH receptor in patients.
Impact 1
Start Year 2006
 
Description Dr Colleen A. Flanagan 
Organisation University of the Witwatersrand
Department Medical School Witwatersrand
Country South Africa 
Sector Academic/University 
PI Contribution We made main contribution to this collaboration.
Collaborator Contribution Co-supervision of a PhD student
Impact 3
 
Description Edward C Hulme 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental design, construction of mutants, writing up
Collaborator Contribution experiemntal design and lab work
Impact 1
 
Description Gray NK 
Organisation Medical Research Council (MRC)
Department MRC Human Reproductive Sciences Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Molecular modelling
Collaborator Contribution 1
Impact 1
Start Year 2011
 
Description Katalin Torok 
Organisation St George's University of London
Department Department of Basic Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We initiated the project regarding the interaction of Kisspeptin receptor (GPR54) with calmodulin and calmodulin kinase II.
Collaborator Contribution The collaborator provided us with some research materials and the approach to detect the interactions between GRP54 and calmodulin/CaMKII.
Impact 0
Start Year 2010
 
Description Stephen O'Rahilly 
Organisation University of Cambridge
Department Institute of Metabolic Science (IMS)
Country United Kingdom 
Sector Academic/University 
PI Contribution Characterization of the mutant receptor in vitro.
Collaborator Contribution Identified a natural mutation of the GnRH receptor in patients.
Impact 1
Start Year 2006
 
Description 9th International Symposium on GnRH: The Hypothalamic-Pituitary-Gonadal Axis in Cancer and Reproduction 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact This symposium brought basic resreacher, clinician and clinic researcher, chemists and pharmacologists around the world under one roof to discuss progress made on GnRH and potential collaboration.

Invited talk.
Year(s) Of Engagement Activity 2008
 
Description Annual Meeting of the Endocrine Scoiety, USA 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact Research conference

To present the work carried out at the MRC HRSU.
Year(s) Of Engagement Activity 2007,2008
 
Description BioScience 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact Research conference

To present reaesrch work carried out at the MRC HRSU.
Year(s) Of Engagement Activity 2006,2007
 
Description British pharmacological society summer meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Invited talk at the BPS summer meeting

Symposium lecture
Year(s) Of Engagement Activity 2009
 
Description Gordon Research Conference in Molecular Pharmacology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact GRC are prestigious conferecenes to discuss research progress within a specific area.

To present the work carried out at the MRC Unit.
Year(s) Of Engagement Activity 2007,2009
 
Description School Visiting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact About 30 researchers and students attended the talk

Scientific inspiration
Year(s) Of Engagement Activity 2010
 
Description Seminars at MRC LMB and NIMR 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact Invited talks at MRC LMB and NIMR.

For potential collaboration.
Year(s) Of Engagement Activity 2007