Xpr1 has been well characterized as a cellular receptor for polytropic and xenotropic MLVs, but its physiological function, aside from that predicted by its homology to the yeast signaling protein Syg1, has not been characterized. Xpr1 is a highly conserved protein with orthologs in animals, plants, and unicellular organisms, indicating its importance in basic cellular functions. This is underscored by the finding that the deletion of Xpr1 in mice is embryonic lethal (Lexicon Pharmaceuticals, personal communication), indicating an important role for Xpr1 in mammals.
Here we demonstrate that hXpr1 regulates cAMP in a manner analogous to that of known GPCRs and that it interacts with the Gβ subunit of the large G-protein complex. While a physiologic ligand for Xpr1 is unknown, we can force signaling through the Xpr1 pathway by the overexpression of Xpr1. Like known Gα-stimulatory GPCRs, Xpr1 activation increases intracellular levels of cAMP, which acts as a second messenger and induces the downstream gene expression of CREB-responsive genes. However, unlike all other previously described GPCRs, Xpr1 contains eight putative transmembrane domains, as opposed to the canonical seven, and Xpr1 shows no sequence similarity to known GPCRs. Despite some similarity of Xpr1 to known phosphate transporters and phosphate transport regulators in yeast, Neurospora, rice, and Arabidopsis, we were unable to demonstrate a role for Xpr1 in phosphate uptake.
Interestingly, the expression of the 200-, 229-, and 248-aa C-terminal truncations of Xpr1, which lack most or all of the Xpr1 transmembrane domains, induced constitutive signaling. We hypothesize that this is due to the strong binding of Gβ by these truncated Xpr1 proteins, with the release of Gα into the cytoplasm, the constitutive activation of adenylate cyclase, and a resulting increase in cAMP levels. These results parallel previously reported findings with 400- and 417-aa C-terminally truncated Syg1 proteins in yeast (34
) and indicate that the N termini of Xpr1 and Syg1 have similar functions despite having markedly different lengths and only 29% amino acid similarity (if multiple gaps in the alignment are ignored) or 17% similarity (if gaps are included).
We found that several retroviruses that utilize Xpr1 as a receptor for cell entry could inhibit Xpr1-mediated cAMP production, resulting in the apoptosis and death of some cell types. Interestingly, XMRV showed the most rapid toxic effect on SY5Y cells, while the MCF 98D virus showed the most rapid toxic effect on Mv1Lu mink lung cells. NZB virus had an intermediate effect on mink cells and had a lower level of toxicity, if any, than XMRV in SY5Y cells. All of these viruses can use the Xpr1 orthologs expressed in SY5Y and mink cells for cell entry. Presumably, the differential toxicity of these viruses is due largely to differences in their interactions with Xpr1 orthologs from different species. However, beyond initial receptor binding by Env, we do not know the mechanism of toxicity, which may involve the downregulation of Xpr1 signaling or the degradation of Xpr1-Env complexes during protein synthesis or following internalization from the cell surface.
While it seems clear now that XMRV is not associated with CFS, our study provides unique insights into a potential role for Xpr1 in neural biology. A role for deregulated cAMP signaling has clear precedents in other disease models and developmental observations. For example, mouse embryos lacking CREB, a major downstream transducer of G-protein signaling, exhibit excess apoptosis and degeneration of sensory and sympathetic neurons, and a CREB knockout is ultimately embryonic lethal (21
). Our studies show that CREB is activated by Xpr1 signaling because CREB is the transcription factor that drives SEAP expression in the assay that we employed to measure cAMP levels. In addition, cAMP-modulating drugs have been shown to reverse neurodegeneration in cultured hippocampal neurons treated with amyloid β-peptide, a model of Alzheimer's disease (37
). Moreover, mice infected with multiple neuropathic polytropic MLVs displayed increased expression levels of proinflammatory cytokines and chemokines, and neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis induce similar cytokine and chemokine expressions (29
Eurexpress Transcriptome Atlas data indicate that brain tissues show the highest Xpr1 expression levels in the mouse (6
), lending support to the hypothesis that Xpr1 is involved in normal brain function. Notably, neuropathology can be induced in mice following infection with various retroviruses, and some studies have shown that polytropic retroviruses are neuropathic, while related ecotropic retroviruses that do not use Xpr1 as a receptor are not (29
). However, neuropathology can also be found in mice following infection by some ecotropic retroviruses, such as CasBr-E MLV, that do not use Xpr1 as a receptor (32
). The infection of mice with ecotropic retroviruses frequently results in recombination events leading to the production of polytropic retroviruses (32
), and it would be interesting to determine if these recombinant viruses play a role in neurologic diseases induced by ecotropic retroviruses.
Our findings demonstrate the critical role which Xpr1 plays in mediating both xenotropic and polytropic retrovirus pathologies and further elucidate the function of Xpr1 in normal cells. However, there remain many questions regarding the normal physiological role of Xpr1 in animals, including what extracellular ligands might modulate Xpr1 activity. While our data indicate that Xpr1 provides a prosurvival signal to the cell, a more specific role for Xpr1 in signal transduction remains to be determined. It will be important to understand whether Xpr1 acts in a fashion analogous to that of other GPCRs and transmits a signal for a particular cellular process or whether it regulates GPCR and cAMP signaling in a more global fashion.