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Lyso-PS (lyso-phosphatidylserine) has been shown to activate the Gi/o-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.

Directed cloning approaches and large-scale sequencing of several vertebrate genomes unveiled many new members of the G-protein-coupled receptor (GPCR) superfamily, among them GPR34. Initial studies showed that GPR34 is an evolutionarily old GPCR structurally related to a group of ADP-like receptors. To gain insight into the genomic organization, regulation of expression, and supragenomic diversification of GPR34 several vertebrate species were analyzed. In contrast to the obviously intronless coding region GPR34 displays an evolutionary preserved 5′ noncoding intron–exon structure. Further, an alternatively used cryptic intron was identified within the coding region, which shortens the N terminus by 47 amino acids. Ubiquitous expression of GPR34 is driven by genomic sequences upstream of at least two transcriptional start regions in mouse and rat but only one region in human. In rodents, both promoters are active in all tissues investigated, but the level of activity is tissue-specific. At the translational level, several conserved in-frame AUGs within the first 150 bp of the coding region may serve as start points for translation in human and other mammals. Combinatory mutagenesis and expression of reporter constructs confirmed these multiple translational start points and revealed a preference for the second in-frame AUG in human GPR34. Our data show that multiple translation initiation starts and alternative splicing contribute to the supragenomic diversification of GPR34.

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.