Phosphoproteomic and functional approaches reveal changes in sperm-specific proteins downstream KOR in human spermatozoa
G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptors superfamily that in response to external stimuli, transduce signals via G proteins to initiate different intracellular signaling pathways which culminate in specific cellular responses. Although in somatic cells the GPCR participate in a broad variety of physiological processes, the expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. Mature spermatozoa could present unique features in their molecular mechanisms downstream GPCRs due to the fact that they possess sperm-specific proteins and are transcriptionally and translationally silent. In order to decipher the signaling pathways engaged by this receptor superfamily in human spermatozoa, we selected the kappa-opioid receptor (KOR) as a study model and applied, for first time, phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses combined with functional studies using a specific agonist of KOR, U50488H.
Sample Processing Protocol
Human spermatozoa that had normal parameters were treated with 1 μM U50488H (the specific agonist of the receptor) for 1 and 60 minutes independently for proteomic and functional analyses. For proteomic analyses, we adopted a Tandem Mass-Tag (TMT) 6-plex isotopic labeling strategy followed by phosphopeptide enrichment by consecutive incubations with titanium dioxide beads (TiO2) and generated samples for LC-MS/MS. The experiment was performed in three biological replicates.
Data Processing Protocol
Peptide and protein searches were performed using the Andromeda search engine (integrated in MaxQuant, version 220.127.116.11) at a FDR threshold of 1%. Perseus software (v.18.104.22.168) was employed for the calculation of the statistical significance (two-sample student´s T-test) and fold changes between U50488H-treated and Control samples. We considered as U50488H-dependent phosphosites the ones that were consistently regulated presenting a 1.5 fold change (U50488H/Ctr > 1.5 or U50488H/Ctr < 0.67 and p value 0.05) in the three replicas for each timepoint and protein fraction.
Itziar Urizar Arenaza, University of Basque Country (UPV/EHU)
Irina Kratchmarova, Department of Biochemistry and Molecular Biology Campusvej 55 5230 Odense M Denmark email@example.com Phone: +45 65502494 ( lab head )
Urizar-Arenaza I, Osinalde N, Akimov V, Puglia M, Candenas L, Pinto FM, Muñoa-Hoyos I, Gianzo M, Matorras R, Irazusta J, Blagoev B, Subiran N, Kratchmarova I. Phosphoproteomic and functional analyses reveal sperm-specific protein changes downstream of kappa opioid receptor in human spermatozoa. Mol Cell Proteomics. 2019 PubMed: 30622161