Poster Presentation International Peptide Symposium 2023

Constitutive activity of the ghrelin receptor provides a dominant second-messenger switch to re-code dopamine D2 receptor output required for co-ordination of voluntary defecation. (#239)

Sebastian G.B. Furness 1 2 , Mitchell T Ringuet 3 , Farhad Dehkhoda 1 , Emily A Whitfield 2 , Linda J Fothergill 4 , Desye Misgenaw 1 , John B Furness 3
  1. The University of Queensland, St. Lucia, QLD, Australia
  2. Drug Discovery Biology, Monash Institute of Pharmaceutical Sceinces, Parkville, Victoria, Australia
  3. Florey Institute of Neuroscience and Mental Health, Parkville, Vic, Australia
  4. Walter and Eliza Hall Institute, Parkville, Victoria, Australia

The dopamine D2 (DRD2) and ghrelin (GHSR) receptors are co-expressed in preganglionic neurons of the spinal defecation centre.  Pharmacological stimulation of either receptor results in colonic pressure waves, however ghrelin is absent from the central nervous system.  In most neurons, activation of DRD2 results in neuronal inhibition via Gαi/o and subsequent G protein-gated inwardly rectifying potassium channel activation.  Contrastingly, DRD2 activation in defecation centre neurons results in depolarisation.  This cellular output switch has been reported in hypothalamic neurons co-expressing GHSR with DRD2 and was previously attributed to heterodimerisation of these receptors.

In spinal defecation neurons, those exhibiting an excitatory response to dopamine also exhibited an excitatory response to GHSR agonists.  Inverse agonists of GHSR blocked DRD2 dependent excitatory response, which was also dependent on intracellular calcium stores.  This switch in DRD2 coupling to calcium mobilisation was also observed in recombinant cells, however this does not appear to be the result of a switch in the G protein preference of DRD2.  Moreover, we were unable to detect allostery between these receptors using ligand binding, nor did we observe them to be close enough to indicate dimerization using super-resolution microscopy.  Instead, we observed a GHSR dependent priming of phospholipase C beta (PLC-b), that was dependent on GHSR’s constitutive activity.  In cells co-expressing a GHSR polymorph that lacks constitutive activity, coupling of DRD2 to calcium was restored by priming with a low concentration ghrelin.  The requirement for PLC-b was also seen in spinal defecation neurons, where PLC-b inhibition reversed the response of these cells to dopamine from excitatory to inhibitory.  Together this data indicates that dopamine mediated excitation is dependent on GHSR constitutive activity via a dominant second-messenger switch.  This work has broad implications for determining metabotropic neurotransmitter responses via modulation through other G protein-coupled receptors.