Abstract: The aim of this study was to screen the interaction proteins and receptor of CART (cocaine and amphetamine regulated transcript peptide), which was a key negative regulatory protein of follicular development in bovine. The both ovaries of 3 healthy cows were collected, after sepa-rating follicles, the GCs (granulosa cells) were scraped and mixed. The membrane proteins were extracted by transmembrane protein extraction kit. The protein G agarose magnetic beads were used to incubate with recombinant CART protein and Anti-CART antibody (n=3). CART-protein complex was eluted and analyzed by LC-MS/MS. The obtained proteins were filtered, and function cluster was analyzed by bioinformatics technology, subcellular localization and transmembrane region analysis of these proteins were performed by CELLO V2.5 software. The intracellular and extracellular structural segments of GPCRs (G-protein-coupled receptors) with 7 times transmembrane were obtained and analyzed by ProtScale software. The GPCRs were used as candidate receptors of CART, their structural characteristics were consistent with the structural characteristics of neuropeptide and receptor binding. The 149, 557 and 298 interaction proteins were identified by immunoaffinity chromatography (n=3), respectively, and total 620 proteins were identified after screening and elimating repetitive proteins. Functional enrichment analysis of multiple database sets showed that GNAS, GNG8 and JUP involved in signal transduction activity, HSD3B involved in steroid biosynthesis, OPA1 and S100A11 involved in regulation of cell apoptosis or proliferation, and ERH involved in Notch signaling pathway, they were interaction proteins and closely related to follicular development in bovine. A total of 156 membrane proteins were obtained by subcellular localization and transmembrane domain analysis, which was 20.53% of total protein. Out of 156, 8 proteins with 7 times transmembrane were considered as GPCRs. The TMHMM analysis showed that the characteristics of ZMPSTE24, HM13 and GPR108 was consistent with the structural characteristics of neuropeptide receptor, their N-terminal was outside and C-terminal was inside. The analysis of PredictProtein binding site showed that there was a G-protein binding site in the intracellular loop of 5-6 transmembrane region of ZMPSTE24. As a candidate receptor for CART, ZMPSTE24 needs to be further verified by molecular interaction and functional tests. This study has laid a foundation for the identification of CART receptors in bovine follicles, and was of great significance to enrich the theory of regulation of follicular development in bovine.

Key words: bovine, follicle, CART, GPCRs, mass spectrometry