Novel protein interaction network of human calcitonin receptor-like receptor revealed by label-free quantitative proteomics

Abstract

AbstractBackgroundG protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) signalling is implicated in skin-related and cardiovascular diseases, migraine and cancer. However, beyond its agonists and receptor activity-modifying proteins (RAMPs), proteins which bind to CLR and define its properties in primary human cells remain insufficiently understood.AimWe aimed to profile the CLR interactome in primary human dermal lymphatic endothelial cells (HDLEC), where this GPCR is expressed.Materials and methodsImmunoprecipitation (IP) of core- and terminally-glycosylated CLR from primaryin vitrocultured HDLEC was conducted using rabbit polyclonal anti-human CLR serum (with pre- immune serum serving as a control) and confirmed by immunoblotting. Total HDLEC and co-immunoprecipitated CLR proteomes were analysed by label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). Quantitativein-situproximity ligation assay (PLA) using ZEISS LSM 710 confocal microscope and ZEN Blue 3.0 and Image J software was performed to confirm nLC-MS/MS findings. All experiments were repeated at least three times (biological replicates). For statistical analysis of PLA data, distribution was analysed using Shapiro-Wilk normality test followed by an unpairedt-test or Mann-Whitney test with ap-value of ≤0.05 interpreted as significant. For MS data of CLR IP samples, statistical analysis was performed usingt-test with a permutation-based false discovery rate (FDR)-adjustedp-value of ≤0.006 interpreted as significant.ResultsA total of 4,902 proteins were identified and quantified by nLC-MS/MS in primary HDLEC and 46 were co-immunoprecipitated with CLR (p<0.006). Direct interaction with the GPCR was confirmed for five of these by PLA (p<0.01).ConclusionsThis is the first study of its kind to identify novel binding partners of CLR expressed in primary human cells. Our integrative quantitative approach, combining immunoprecipitation of core- and terminally-glycosylated CLR, nLC-MS/MS, and PLA, could be applied in a similar fashion to study its interactome in a variety of human cells and tissues, and its contribution to a range of diseases, where the role of this GPCR is implicated.