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

Manolis, Dimitrios; Hasan, Shirin; Ettelaie, Camille; Maraveyas, Anthony; O'Brien, Darragh; Kessler, Benedikt; Kramer, Holger; Nikitenko, Leonid


Dimitrios Manolis

Shirin Hasan

Camille Ettelaie

Darragh O'Brien

Benedikt Kessler

Holger Kramer


G 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.
We aimed to profile the CLR interactome in primary human dermal lymphatic endothelial cells (HDLEC), where this GPCR is expressed.
Materials and methods
Immunoprecipitation (IP) of core- and terminally-glycosylated CLR from primary in vitro cultured 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). Quantitative in-situ proximity 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 unpaired t-test or Mann-Whitney test with a p-value of ≤0.05 interpreted as significant. For MS data of CLR IP samples, statistical analysis was performed using t-test with a permutation-based false discovery rate (FDR)-adjusted p-value of ≤0.006 interpreted as significant.
A 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).
This 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.


Manolis, D., Hasan, S., Ettelaie, C., Maraveyas, A., O'Brien, D., Kessler, B., …Nikitenko, L. Novel protein interaction network of human calcitonin receptor-like receptor revealed by label-free quantitative proteomics

Deposit Date Apr 24, 2023
Publicly Available Date Apr 25, 2023
Keywords endothelial cell, lymphatic, proteomics
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Additional Information This article is a preprint and has not been peer-reviewed. It reports new medical research that has yet to be evaluated and so should not be used to guide clinical practice.


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