A seemingly universal new family of receptors, the PAQRs, that originated from ancestral bacterial hemolysin encoding genes has been described in eukaryotes [7
]. Much controversy surrounds these receptors specifically, their membrane topology and the possibility of being coupled to G protein signalling pathways [17
]. Nevertheless, the nature of the ligands bound by a particular receptor has been solved for most PAQRs. They have been observed to bind either the peptide hormone adiponectin or the steroid hormone progesterone [38
]. This brings up another consideration; should the adiponectin and progesterone receptors be considered members of the same family of receptors? The fact that the nature of the ligands are so different, together with the differences observed in membrane topology between the Class I (adiponectin receptors) and Class II (progesterone receptors) suggest inherent difference between the two classes. Class I receptors have been predicted to have the N-terminal in the interior of the cell while Class II receptors have the usual GPCR topology of the N-terminal outside of the cell and the C-terminal inside the cell [8
]. Due to the predicted membrane topology of the progesterone receptors, it is suggests that they might be a new class of GPCRs. In this paper we report a new member of the Class II PAQRs and address the issues regarding membrane topology, ligand binding and its relationship to the S. schenckii
G alpha subunit SSG-2, in an effort to characterize the SsPAQR1.
The fact that SsPAQR1 was identified in a Y2H assay with a G protein alpha subunit as bait, offers for the first time direct evidence of the association of these receptors to the heterotrimeric G protein signalling pathways. This association was verified using Co-IP. Indirect evidence of the association of progesterone PAQRs to G proteins has been reported by other investigators. One of these instances involves fish oocyte maturation where response to a novel progesterone hormone was associated to a pertussis-sensitive Gαi subunit pathway [6
Transmembrane analysis of the SsPAQR1 described here predicts that this protein has the 7 transmembrane domains characteristic of GPCRs like other progesterone binding members of the PAQR family. The bioinformatic analyses described above (TMHMM, SOUSI and MEMSAT-SVM) predicted that the N-terminal region is localized outside the plasma membrane while the C-terminal region is intracellular. This orientation has also been observed in progestin receptors, PAQR6 and mPRa [6
]. In the case of the adiponectin members of the PAQR family such as the human adiponectin receptor 2 and 3, the orientation seems to be the opposite, as stated previously [12
Bioinformatic analyses also show that SsPAQR1 and its fungal homologues from M. oryzae
, T. reesei
, N. crassa
and P. anserina
, among others belong to the PAQR receptor family. These homologues exhibit approximately 65 to 80% identity to SsPAQR1. The transmembrane domain analyses of some of these fungal homologues showed that most have the 7 transmembrane domains characteristic of the GPCRs. TMHMM analysis also shows that they have the traditional orientation of an external N-terminal domain and an internal C-terminal domain as SsPAQR1, except in the case of Izh3 where the N-terminal is inside and the C-terminal is outside (Additional file 2
). It is also of interest to note that in many filamentous and dimorphic fungi where whole genomes have been sequenced, the SsPAQR1 homologues encode proteins of approximately 500 amino acids in addition to at least one more Izh homologue of smaller size (approximately 300 amino acids).
Steroid binding proteins have been described for various yeasts [42
]. Many studies have predicted the existence of a progesterone receptor in the membrane of filamentous fungi such as Rhizopus nigricans
] but the molecular basis of steroid signalling in fungi remains unresolved [43
]. Progesterone has been reported to bind to enriched plasma membrane fractions of R. nigricans
with high affinity and this hormone has been reported to induce an activation of G proteins that decreases in the presence of cholera toxin [29
]. Nevertheless, to date no progesterone receptor has been directly identified in this or any other fungi. This work identified a membrane progesterone receptor for the first time in fungi. Progesterone was identified as the ligand corresponding to SsPAQR1 using the yeast-based assay [23
]. This assay was used previously to identify the ligands of human PAQRs heterologously expressed in S. cerevisae
]. This assay is specific for PAQRs and was intended for the study of these receptors without the intervention of other possible progesterone binding protein. Using this assay, SsPAQR1 was expressed in S. cerevisiae
and progesterone was identified as the ligand for SsPAQR1. Yeasts carrying the empty expression vector showed that progesterone did not affect FET3
, showing that the effect was not due to a nonspecific effect of progestrone on S. cerevisiae.
Progesterone responsiveness was only observed if SsPAQR1 was being expressed. These results put an end to the uncertainty regarding the presence of a membrane progesterone receptor in fungi.
However, the question as to why fungi have a steroid hormone receptor remains unanswered. The effects of progesterone and other steroids on fungi have not been fully documented. In Candida albicans
the response to steroid hormones leads to the activation of transcription of genes encoding the ATP-binding cassette of drug efflux pumps [47
]. In S. cerevisiae
exposure to progesterone results in the up-regulation of stress response genes such as those involved in transport, oxidative stress response, growth, cell division and cell wall biogenesis, among other [43
In the filamentous fungi, most of the information regarding progesterone and fungi is related to bioconversion of the different steroid metabolites by fungi. Recently, a progesterone-hydroxylating enzyme system was studied and found to be dependent on the G protein beta subunit and cAMP in Fusarium oxysporum
]. The authors proposed that progesterone is toxic to this fungus and that by the induction of the enzymes involved in the hydroxylation of progesterone, the fungus is able to reduce the toxicity associated with the hormone. This transformation results in a more soluble compound that can be excreted to the medium.
The toxicity of progesterone results in an inhibition of growth in R. nigricans
]. This inhibition of growth was explained as resulting from a decrease in cAMP caused by progesterone. In this work we also report an inhibition of growth of both the mycelium and yeast forms of the fungus in the presence of progesterone, the yeast form being the most affected. Nevertheless, we could not correlate this inhibition of growth to a decrease in cAMP concentrations.
Another major area of concern regarding progesterone PAQRs is the determination of the specific signal generated upon the interaction of the receptor with its ligand. Different theories have suggested that cAMP and/or calcium could be involved. Nevertheless, even in situations where adenylate cyclase has been identified as a target of the possible effects of progesterone, there is still disagreement if the hormone causes a decrease or an increase in cAMP, and the time considered reasonable for the effect on this cyclic nucleotide to be observed [50
]. The addition of progesterone to S. schenckii
yeast cells prior to harvesting for cAMP determinations showed that the levels of intracellular cAMP increased during the first minute after exposure to the ligand and decreased significantly after five hours incubation with the hormone. The increase in the cytosolic concentration of cAMP could be the result of the interaction of the ligand and the receptor resulting in the activation of SSG-2 that in turn triggers the cascade of events leading to an increase in cAMP. The response to the ligand in steroid membrane receptors has been identified as occurring in 1 to 5 min in the case of sperm motility to up to 6-18 h in the case of oocyte maturation experiments [50
The work reported here identifies the presence of a progesterone receptor in S. schenckii
for the first time and establishes the presence of homologous of this receptor in other fungi as well. Other authors who studied the response of fungi to progesterone have proposed the existence of this receptor. Although the question still remains regarding the benefit of having such receptors in fungal cells remains open, one could argue that fungi are in contact with plant and other fungal steroids in their environment and that they have the capacity to transform these molecules to suite their needs [52