Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants.
OBJECTIVE
In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms.


METHODS
Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [(35)S]-GTPgammaS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC(4) and LTD(4). Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively.


RESULTS
The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC(50). Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Galpha(i).


CONCLUSIONS
We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.
Department of Neuroscience and Pharmacology, The Panum Institute, Copenhagen University, Denmark.
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Benned-Jensen
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Rosenkilde
D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000378:metabolism; D015289:Leukotrienes / Q000378:metabolism; D008024:Ligands; D009206:Myocardium / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C121670:GPR17 protein, human; D015289:Leukotrienes; D008024:Ligands; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled