Quantitative proteomics reveals CLR interactome in primary human cells.

Area of Science:

• Cellular Biology
• Molecular Biology
• Proteomics

Background:

• The calcitonin receptor-like receptor (CLR), a G protein-coupled receptor (GPCR), plays crucial roles in various cell types and is linked to diseases like cancer and cardiovascular pathologies.
• The protein interaction network of CLR (CLR interactome) in primary cells, expressing physiologically relevant levels of the receptor, has not been previously characterized.
• Understanding the CLR interactome is essential for deciphering its biological functions and pathological implications.

Purpose of the Study:

• To comprehensively identify and characterize the proteome-wide interactome of endogenously expressed CLR in primary human cells.
• To establish a novel integrative methodological workflow for analyzing GPCR interactomes at endogenous levels.
• To uncover previously unreported protein interactions with CLR.

Main Methods:

• Utilized primary human dermal lymphatic endothelial cells.
• Employed an integrative approach combining immunoprecipitation with anti-human CLR antibody, label-free quantitative nano LC-MS/MS, and quantitative in situ proximity ligation assay.
• Performed proteome-wide analysis to identify CLR-interacting proteins.

Main Results:

• Identified 37 novel proteins interacting with endogenously expressed CLR out of 4902 detected proteome members.
• Revealed direct interactions between CLR and two kinases and two transporters using proximity ligation assays.
• None of the identified interactors were previously reported in the CLR interactome.

Conclusions:

• The study unveils the previously unrecognized complexity of the CLR interactome in primary cells.
• The findings provide fundamental insights into the biology of CLR and its interactions.
• The developed integrative methodology and generated datasets serve as a valuable resource for future research on CLR interactome in various cell types and diseases.