Parathyroid hormone (PTH) secretion maintains free-ionised extracellular calcium (Ca2+o) homeostasis under the control of the calcium-sensing receptor (CaR). In humans and dogs, blood acidosis and alkalosis is associated with increased or suppressed PTH secretion respectively. Furthermore, large (1.0 pH unit) changes in extracellular pH (pHo) alter Ca2+o sensitivity of the CaR in CaR-transfected HEK-293 cells (CaR-HEK). Indeed, it has been found in this laboratory that even pathophysiological acidosis (pH 7.2) renders CaR less sensitive to Ca2+o while pathophysiological alkalosis (pH 7.6) increases its Ca2+o sensitivity, both in CaR-HEK and parathyroid cells. If true in vivo, then CaR’s pHo sensitivity might represent a mechanistic link between metabolic acidosis and hyperparathyroidism in ageing and renal disease. However, in acidosis one might speculate that the additional H+ could displace Ca2+ bound to plasma albumin, thus increasing free-Ca2+ concentration and so compensating for the decreased CaR responsiveness. Therefore, I first demonstrated that a physiologically-relevant concentration of albumin (5% w/v) failed to overcome the inhibitory effect of pH 7.2 or stimulatory effect of pH 7.6 on CaR-induced intracellular Ca2+ (Ca2+i) mobilisation. Determining the molecular basis of CaR pHo sensitivity would help explain cationic activation of CaR and permit the generation of experimental CaR models that specifically lack pHo sensitivity. With extracellular histidine and free cysteine residues the most likely candidates for pHo sensing (given their sidechains’ pK values), all 17 such CaR residues were mutated to non-ionisable residues. However, none of the resulting CaR mutants exhibited significantly decreased CaR pHo sensitivity. Even co-mutation of the two residues whose individual mutation appeared to elicit modest reductions (CaRH429V and CaRH495V) failed to exhibit any change in CaR pHo sensitivity. I conclude therefore, that neither extracellular histidine nor free cysteine residues account for CaR pHo sensitivity. Next, it is known that cytosolic cAMP drives PTH secretion in vivo and that cAMP potentiates Ca2+o-induced Ca2+i mobilisation in CaR-HEK cells. Given the physiological importance of tightly controlled PTH secretion and Ca2+o homeostasis, here I investigated the influence of cAMP on CaR signalling in CaR-HEK cells. Agents that increase cytosolic cAMP levels such as forskolin and isoproterenol potentiated Ca2+o-induced Ca2+i mobilisation and lowered the Ca2+o threshold for Ca2+i mobilisation. Indeed, forskolin lowered the EC50 for Ca2+o on CaR (2.3 ± 0.1 vs. 3.0 ± 0.1 mM control, P<0.001). Forskolin also potentiated CaR-induced ERK phosphorylation; however protein kinase A activation appeared uninvolved in any of these effects. Pertussis toxin, used to block CaR-induced suppression of cAMP accumulation, also lowered the Ca2+o threshold for Ca2+i mobilisation though appeared to do so by increasing efficacy (Emax). Furthermore, mutation of the CaR’s two putative PKA consensus sequences (CaRS899 and CaRS900) to a non-phosphorylatable residue (alanine) failed to alter the potency of Ca2+o for CaR or attenuate the forskolin response. In contrast, phosphomimetic mutation of CaRS899 (to aspartate) did increase CaR sensitivity to Ca2+o. Together this suggests that PKA-mediated CaRS899 phosphorylation could potentiate CaR activity but that this does not occur following Ca2+o treatment in CaR-HEK cells. Together, these data show that cAMP regulates the Ca2+o threshold for Ca2+i mobilisation, thus helping to explain differential efficacy between CaR downstream signals. If true in vivo, this could help explain how multiple physiological signal inputs may be integrated in parathyroid cells.

Les récepteurs couplés aux protéines G (RCPG) démontrent de plus en plus de capacités à activer des mécanismes jusqu’alors associés à des facteurs de transcription ou des molécules d’adhésion. En effet, de nouvelles preuves rapportent qu’ils pourraient également participer au guidage axonal qui est le mécanisme permettant aux axones de cellules nerveuses de rejoindre leur cible anatomique. Le guidage axonal se fait par l’interaction entre les molécules de guidage et une structure particulière présente à l’extrémité de l’axone, le cône de croissance. Par exemple, les RCPGs participent au guidage des cellules ganglionnaires de la rétine (CGR), dont les axones s’étendent de la rétine jusqu’au noyaux cérébraux associés à la vision. Cet effet est observé avec des RCPGs tels que les récepteurs aux cannabinoïdes (CB1 et CB2) et celui du lysophosphatidylinositol, le GPR55. Les RCPGs GPR91 et GPRG99, respectivement récepteurs au succinate et à l’α-cétoglutarate, se trouvent à la surface de ces CGRs, ce qui en font des candidats potentiels pouvant participer au guidage axonal. Dans ce mémoire, l’effet des ligands de ces récepteurs sur la croissance et la navigation des axones des CGRs fut analysé. L’impact produit par ces récepteurs ainsi que leurs ligands sur la morphologie des cônes de croissance fut déterminé en mesurant leur taille et le nombre de filopodes présents sur ces cônes. Pour évaluer le rôle du succinate et de l’a-cétoglutarate sur la croissance globale des axones de CGRs, la longueur totale des projections axonales d’explants rétiniens a été mesurée. L’effet de ces ligands des récepteurs GPR91 et GPR99 sur le guidage axonal a également été évalué en temps réel à l’aide d’un gradient créé par un micro injecteur placé à 45° et à 100µm du cône de croissance. La distribution in vivo des récepteurs GPR91 et GPR99 sur la rétine a été étudié à l’aide d’expériences d’immunohistochimie. Les résultats obtenus indiquent que l’ajout de 100µM de succinate produit une augmentation de la taille des cônes de croissance et du nombre de filopodes présents à leur surface. Il augmente également la croissance des axones. Ce type de réponse fut également observé lorsque les cellules furent soumises à 200µM d’α-cétoglutarate. Fait à noter, les deux récepteurs n’ont pas d’impact sur le guidage axonal. Ces résultats indiquent donc que les agonistes des récepteurs GPR91 et GPR99 augmentent la croissance des cellules ganglionnaires lorsqu’ils sont présents lors du développement. Par contre, ils n’ont pas d’influence sur la direction prise par les cônes de croissance. Ces nouvelles données sont un pas de plus dans la compréhension des mécanismes qui gèrent et participent au développement et la croissance des CGRs, ce qui pourrait donner de nouvelles cibles thérapeutique pouvant mener à la régénération de nerfs optiques endommagés. / G Protein-Coupled Receptors (GPCRs) show a greater role in activating mechanism usually associated to transcription factors or cell adhesion molecules. New evidence shows that some of these receptors have an impact on axon guidance, the mechanism by which neurons’ axons are able to grow from their origin and reach their anatomical target. Guidance is mediated via a structure at the tip of the axon called the growth cone, which can interact with surrounding molecules. Axons from retinal ganglion cells (RCGs) navigating from the retina to the cerebral nuclei associated with vision, are sensitive to some of the GPCR ligands. These GPCRs are the cannabinoid receptors CB1 and CB2 and the GPR55, a receptor for lysophosphatidylinositol. GPR91 and GPR99, respectively receptors for succinate and α-ketoglutarate, are expressed in RGCs making them prime candidates to have an impact on axon guidance. In this thesis, we will test the role of GPR91 and GPR99 ligands on RCG axon growth and guidance. To assess the impact of these receptors and their ligands on axon growth and guidance, first we evaluated their effects on growth cone morphology. To achieve this, we measured growth cone size and filopodia numbers, when exposed to 100 µM of succinate or 200µM of α-ketoglutarate. The effects of these ligands on axon growth were evaluated by measuring the total axon outgrowth from retinal explants. The role of GPR91 and GPR99 on growth cone turning, was determined by exposing a growing axon to a gradient of these ligands, originating from a micropipette situated 100 µm and 45° from the growth cone. The expression of these receptors in the retina was evaluated using immunohistochemistry. Results showed that the addition of 100 µM succinate induced an increase in both growth cone size and filopodia number. It also increased total axon growth. α-ketoglutarate, at a concentration of 200 µM, produced similar results. Noteworthy, both ligands had no effect on growth cone turning. In brief, these results indicate that GPR91 and GPR99 agonists induce an increase in RGC growth when present during development. They, however, have no effects on RGC growth cone turning. These new data provide a better understanding of the mechanisms controlling RGC development.

G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.

G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.