Comments by Dr. Nicola J. Smith, National Heart Foundation Future Leader Fellow & Group Leader, Molecular Pharmacology Laboratory, Victor Chang Cardiac Research Institute, Australia
As is often the case with orphan GPCRs, assigning the endogenous ligand has been controversial for the closely related peptide family orphans, GPR37 and GPR37L1. In 2013, Randy Hall and his team (PubMed: 23690594) first reported an association between both centrally-expressed orphan GPCRs and prosaposin (PSAP) and prosaptide (TX14A), the synthetic active epitope of PSAP. Since that time there has been much debate in the field about whether this pairing is correct, with some authors corroborating the findings (PubMed: 24371137; 30010619, 28795439) and others not (PubMed: 23396314; 27072655; 28688853). Note that Head Activator, found in Hydra, was earlier reported as a ligand (PubMed: 16443751) but was quickly discredited (PubMed:28688853; 23686350).
A recent paper by Sergey Kasparov’s laboratory in Bristol has added further fuel to the fire. In a series of well controlled experiments, Liu et al. (PubMed: 30260505) provided convincing evidence that prosaptide is cyto- and neuro-protective and promotes chemotaxis. They are also the first group to demonstrate an effect of prosaptide at a more physiologically plausible potency. At the same time, Bang et al. (PubMed: 30010619) published a ground-breaking paper linking GPR37 expression to macrophage function. Moreover, they proposed a second, more potent ligand for GPR37 (GPR37L1 was not studied): the pro-resolving mediator neuroprotectin D1 (NPD1). Using HEK293 cells expressing GPR37, NPD1 was a potent stimulator of Gαi/o-dependent calcium flux; findings that were corroborated in macrophages isolated from wild type, but not GPR37 knock-out, mice (PubMed: 30010619). Thus, it may be that the endogenous ligand for GPR37 (and perhaps GPR37L1?) is not a peptide after all, but a lipid.
These two studies, while exciting, do little to help us resolve the conundrum that is PSAP/prosaptide and GPR37/GPR37L1. At the very least, it seems likely that prosaptide, if not the highest affinity endogenous ligand at GPR37, is certainly capable of signalling through GPR37 to stimulate Gαi/o signal transduction (whether it is the most potent endogenous agonist will be shown in time as independent groups seek to validate the actions of NPD1).
But what of GPR37L1? This is harder to answer as a number of studies linking GPR37L1 to PSAP/prosaptide have been performed in double GPR37/GPR37L1 knock-out backgrounds or inappropriate tissue models. For example, in the original paper connecting prosaptide to the receptors, the authors claimed prosaptide acted through both GPR37 and GPR37L1 in primary astrocytes, despite the fact that their Western blots demonstrated marked GPR37 expression in comparison to GPR37L1 in the cells (PubMed: 23690594). More recently, they failed to recapitulate this initial pairing in a HEK293 model (PubMed: 28688853). The absence of GPR37L1 in primary astrocytes is consistent with the animal knock-out work of Marazziti et al. (PubMed: 24062445), who showed that GPR37L1 protein was barely detectable before post-natal day 15, which is after the window for isolating primary astrocyte cultures (confirmed by PubMed: 28795439). Coleman et al. (PubMed: 27072655) overcame this expression issue, with difficulty, by using cerebellar slice cultures in vitro to examine Gαs, but not prosaptide, signalling in wild type and knock-out tissue.
In the neuroprotection paper by Liu et al. (PubMed: 30260505) it is clear that prosaptide or PSAP are exhibiting an effect on the cells. By depleting astrocytes of PSAP and then reintroducing prosaptide exogenously, there is an obvious effect on cell migration, cytotoxicity and neuroprotection – phenotypes that are all lost when shRNA knocking down expression of both GPR37 and GPR37L1 are used. Frustratingly, though, the use of a double knock-down approach makes it impossible to ascribe a specific effect to GPR37L1. While GPR37 and GPR37L1 are very closely related by phylogeny and have highly similsar binding sites (PubMed: 27992882), this does not mean that prosaptide is a ligand at both receptors, nor that both receptors signal via the same G proteins (another area of controversy for GPR37L1, where contradictory studies including two by the same team show either Gαi/o or Gαs signaling: PubMed: 23690594, 30260505 vs 27072655, 28688853). Thus, the failure to use single receptor knock-out/knock-downs, or isolate cells with endogenous expression of GPR37L1, represent major limitations in these studies.
Other than the confounding effects of both GPR37 and GPR37L1 deletion in tested cells, what are other reasons that could explain the inconsistencies between studies? Kasparov and colleagues (PubMed: 30260505) attribute this to cellular background, stating that previous studies that failed to confirm prosaptide/GPR37L1 coupling (PubMed: 27072655, 28688853) used HEK293 cells that must be lacking in the necessary endogenous machinery for signal transduction (PubMed: 30260505). To support this claim, they turned to the PRESTO-Tango assay in HEK293 cells to demonstrate prosaptide stimulation could not lead to GPR37L1-dependent recruitment of beta-arrestin. The assay choice is surprising because previous beta-arrestin-based screens at GPR37L1 have failed to show that the receptor can indeed recruit arrestins (PubMed: 23396314, 25895059), and Liu et al. (PubMed: 30260505) did not provide evidence that recruitment was intact in a more physiologically relevant cellular background. Most puzzling though is that the original paper that identified prosaptide and PSAP as GPR37/37L1 ligands used HEK293 cells to make the original pairing (PubMed: 23690594). They also refute the physiological relevance of high constitutive Gαs signalling reported by Coleman et al., even though Coleman et al. demonstrated higher cAMP accumulation in cerebellar brain slices from wild type mice when compared to GPR37L1-/- (PubMed: 27072655).
Further muddying the waters, the physiological role of GPR37L1 itself remains enigmatic. For example, Min et al. (PubMed: 20100464) initially reported GPR37L1 null mice to have a staggering 62 mmHg increase in systolic blood pressure when compared to a cardiac-specific overexpressing model, with the presence of concomitant left ventricular hypertrophy. However, Coleman et al. (PubMed: 29625592) found a far more marginal cardiovascular phenotype, with a small increase in blood pressure evident in female mice only. Notably, male GPR37L1 knock-out mice appeared to be more susceptible to cardiovascular stressors, while females were cardioprotected (PubMed: 29625592). In terms of a developmental phenotype, Marazziti et al. (PubMed: 24062445) found that GPR37L1 null mice displayed precocious cerebellar development with enhanced performance in a rotarod test up to 1 year of age. More recently, though, Jolly et al. (PubMed: 28795439) failed to confirm a behavioural difference in their own GPR37L1 knock out mice. The links between GPR37L1 and neurological defects are also confounded by the fact that GPR37 also needs to be deleted in mice for a clear phenotype to be evident. For example, while a single point mutation in GPR37L1 (K349N) in a highly consanguineous family appeared to be causative of fatal progressive myoclonus epilepsy, the mouse phenotype was most pronounced in double GPR37/GPR37L1 knock-out animals (PubMed: 28688853). In vitro studies of the GPR37L1 K349N mutant found no difference between it and the wild type receptor in terms of receptor expression, processing, signalling or ubiquitination (PubMed: 28688853). In the absence of a transgenic K349N mutant mouse, or any confirmed synthetic agonists or antagonists, the authors then assessed seizure susceptibility in knock-out mice of either GPR37L1, GPR37 or both receptors. Interestingly, using the 6Hz-induced seizure model, the GPR37-/- mice appeared to have a more pronounced phenotype than the GPR37L1-/- mice, while double KO mice were extremely susceptible to seizures at all frequencies tested. GPR37 and double KO mice both displayed more spontaneous seizures, although curiously in the flurothyl-induced seizure model only GPR37L1-/- differed from wild type. Thus, conclusive links between GPR37L1 and a specific physiological or pathophysiological state remain to be provided and it seems in general that we are far from understanding the true biology and pharmacology of the receptor.