Cloning, Analyzing And Ligand Screening Of HGPCRc As A Member Of Human Orphan G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) are the largest and most diverse group of transmembrane proteins involved in signal transduction. They have been playing key roles in drug discovery and occupying 40% in drug market of new drugs as targets by estimation. More and more orphan GPCRs (oGPCRs), whose endogenous ligands and functions are still to be identified, have been discovered in recent years. For oGPCRs, it is the most important and hard task to find their specific ligands. Once their ligands are identified, they will become novel research areas. It is obvious that oGPCRs have been the most important targets for innovating drugs.In this study, with the aid of the information from gene bank, the whole ORF of a member of oGPCR (AB083598) was amplified by RT-PCR from human colon tissue, designated hGPCRc. Also, the ORF of hGPCRc could be obtained with PCR from the template of genomic DNA of a health volunteer's blood. The results indicated that hGPCRc has no introns and accords with the characteristic of most GPCRs. Software analysis at the NCBI website showed that hGPCRc was localized at 13q32.2, and its corresponding amino acids formed seven-transmembrane domains and had 85% homology with the counterparts of rat or mouse, but a lower homology with other human genes. An unrooted phylogenetic tree drawing from hGPCRc and its related sequences showed that P2Y1 receptor is the member having the highest homology with hGPCRc but only reach 36%. It was indicated that hGPCRc should be a new member of human oGPCRs.For more information on the hGPCRc, the expression profiling of hGPCRc was detected by RT-PCR in different human fetal tissues. The result indicated hGPCRc was expressed dominantly in the heart, kidney and colon etc. It was supposed that hGPCRc might involve in several physiological or pathological processing and serve as potentialtargets in the related diseases. In order to investigate the cellular location of hGPCRc protein, CHO-K1 cells were transfected with the recombinant GFP-hGPCRc and then observed under the laser scanning configal microscopy (LSCM). The result showed that Green fluorescence protein was distributed in whole cell after transfection with pEGFP-N1 plasmid, but only found on membrane after transfection with recombinant GFP-hGPCRc vector. So, hGPCRc was localized on membrane.One of the commonly used strategies to screen ligands is to monitor the second messenger of the engineered cells which can express the protein of oGPCRs. For ligand screening of hGPCRc, a system based on the assaying of [Ca2+]j changes was established according to hGPCRc closing to human P2Y1 receptor which coupled to Gq subunit. To obtain an engineered CHO-hGPCRc cells, CHO-Ki cells were transfected with the recombinant pcDNA3.1(+)-hGPCRc. Fluo-3 was used in assaying the [Ca2+]i changes induced by different compounds in the CHO- hGPCRc cells. Among a dozen of the known compounds which could activate the P2Y1 receptor, no one could activate the hGPCRc. So, hGPCRc might be not a member of the P2Y1 receptors family.In summary, looking for oGPCRs ligands and exploring the physiological functions and pathological meanings of oGPCRs would give a new breakthrougth for innovating drugs study or interpreting the mechanism of related diseases. In the present study, hGPCRc was firstly cloned and analyzed as a member of oGPCRs. Secondly, the expression profiling of hGPCRc was detected and revealed the receptor was dominantly expressed in several tissues including kidney, heart and colon etc. Lastly, base on assaying the [Ca2+]i changes, an engineered CHO-hGPCRc cells were established and used as a system for screening ligand. The screening results indicated that hGPCRc could not be activated by the ligands of P2Y1 and might not belong to P2Y1 family.