Towards a crystal structure of the Family B GPCR PTH2

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

Background: Family B G protein-coupled receptors (GPCRs) are validated drug targets, some already exploited through the development of peptidic drugs (e.g. extenatide at the GLP-1 receptor for type 2 diabetes; teriparatide at PTH1 for osteoporosis). However, the discovery of small molecule ligands for Family GPCRs, particularly agonists, has proven to be intractable. One reason for the lack of success has been the paucity in our understanding of the structure of Family B GPCRs and, in particular, how peptide ligands bind and activate them.
Objectives: (1) To overexpress, purify and crystallise PTH2 using the insect cell/baculovirus system (Saarenpää et al., 2015). (2) To use molecular modelling, site-directed mutagenesis and pharmacological assays: (i) To identify key residues in the 7TM domain that contribute to peptide agonist recognition; (ii) To generate a disulphide-trapped conformational state of PTH2 by cross-linking the peptide ligand to both the NTD and 7TM domains of the full-length receptor in order to stabilise the ligand-bound active state.
Novelty: There are no known structures for any full-length Family B GPCRs, probably due to the extensive conformational flexibility between the N-terminal domain and the 7TM domain - trapping this state using the ligand is a novel approach. The PTH2 receptor is unexplored from a structure-function perspective.
Timeliness: GPCR structure determination is in a golden period but Family B GPCRs are devoid of known structure - progress in this area is important.
Experimental Approach: (1) The insect cell/baculovirus system is already in use in the Goldman laboratory. Virus expressing the PTH2 cDNA will be generated and used for over-expression and purification of PTH2 protein. (2) A molecular model of the full-length peptide-bound PTH2 has already been generated by Donnelly for the planning of site-directed mutagenesis targets. All the necessary pharmacological radio-ligand binding and 384-well cAMP assays are established in Donnelly's lab.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1774805 Studentship BB/M011151/1 30/09/2016 30/03/2021 James Henderson