Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Stabilisation Methods for the Parathyroid Receptor 1 and its Constitutively Active Mutants (#120)

Isabella C Russell 1 2 3 , Xin Zhang 1 2 3 , Matthew Belousoff 1 2 3 , Denise Wootten 1 2 3 , Patrick Sexton 1 2 3
  1. ARC Centre for Cryo-Electron Microscopy of Membrane Proteins , Melbourne, Vic, Australia
  2. Drug Discovery Biology, Melbourne, Vic, Australia
  3. Monash University, Parkville, VIC, Australia

The parathyroid hormone receptor 1 (PTH1R) is a class B1 G protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein, Gαs[1]. Naturally occurring polymorphisms in PTH1R that induce constitutive activity (spontaneous activation of receptor in the absence of an agonist) have been linked to Jansen's metaphyseal chondrodysplasia, a form of short limbed dwarfism; these polymorphisms include H223R, H223K and T410P[2]. These mutations disrupt known polar networks within class B1 GPCRs that are responsible for maintaining the receptor in an inactive confirmation[3]. The expression and Gαs-dependent cAMP signalling of 3 constitutively active PTH1R mutants was assessed using flow cytometry and cAMP accumulation assays. The mutations revealed a scale of increasing constitutive activity, with wild type (WT) PTH1R and H223K being the least constitutively active, followed by T410P and H223R as the most constitutively active. The WT PTH1R, dominant negative Gαs (DNGαs) and Gβ1γ2 complex with the endogenous peptide PTH (1-34) were purified in the presence of the stabilising agent nanobody 35. Negative staining under electron microscope (EM), SDS-PAGE, and western blot revealed the complex was pure and homogenous. The structure of PTH (1-34)-bound WT PTH1R:DNGs:Nb35 complex was solved at the global resolution of 2.41 Å using cryo-EM, with a similar overall conformation as the published structure but higher resolution[4]. This structure will be used as a reference for future studies of the PTH1R mutants:Gαs complexes with and without agonists. This work will provide critical structural insights into the understanding of the constitutive activity of PTH1R.

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  2. Schipani, E., et al., Constitutive activation of the cyclic adenosine 3′, 5′-monophosphate signaling pathway by parathyroid hormone (PTH)/PTH-related peptide receptors mutated at the two loci for Jansen’s metaphyseal chondrodysplasia. Molecular Endocrinology, 1997. 11(7): p. 851-858.
  3. Yin, Y., et al., Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors. Journal of Biological Chemistry, 2017. 292(24): p. 9865-9881.
  4. Kobayashi, K., et al., Endogenous ligand recognition and structural transition of a human PTH receptor. Molecular Cell, 2022.