bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.04.18.537143

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

Manolis, D.·Hasan, S.·Ettelaie, C.·Maraveyas, A.·O'Brien, D. P.·Kessler, B. M.·Kramer, H. B.·Nikitenko, L. L.
View original source ↗Export metadata ↓

Abstract

BackgroundG 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 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 [&le;]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 [&le;]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.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Manolis, D., Hasan, S., Ettelaie, C., Maraveyas, A., O'Brien, D. P., Kessler, B. M., Kramer, H. B., Nikitenko, L. L.. 2023-04-19. Novel protein interaction network of human calcitonin receptor-like receptor revealed by label-free quantitative proteomics. https://doi.org/10.1101/2023.04.18.537143

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A dynamic phosphoproteome reveals stage-specific regulation of cytokinesis in Leishmania

Leishmania parasites undergo extensive morphological and metabolic remodelling during their transition from motile insect promastigotes to non-motile intracellular amastigotes, yet how dynamic protein kinase signalling drives this process remains poorly understood. Here, we present a high-resolution temporal phosphoproteome of L. mexicana promastigote to axenic amastigote differentiation, profiling 16,495 phosphosites on 3,101 proteins across seven developmental time points and identifying 5,795 dynamically regulated sites. This temporal map reveals stage-specific signalling programs, including regulated sites in MPK2 and non-regulated sites in MRK1, protein kinases known to be essential for amastigote differentiation. Using analogue-sensitive (AS) chemical genetics, we show that acute inhibition of either kinase in axenic amastigotes disrupts the spatial coordination of cleavage furrow ingression with nuclear and kinetoplast segregation, revealing essential cytokinesis roles obscured in constitutive gene deletions. Despite generating similar cytokinesis phenotypes, genetic over-expression and cellular localisation analyses demonstrate that MPK2 and MRK1 act through spatially segregated signalling pathways. Time-resolved phosphoproteomics upon MPK2 inhibition places its activity upstream of cytoskeletal remodelling networks. Importantly, we show that the stage-specific requirement for MPK2 is defined by the parasite's morphology: while motile promastigotes tolerate MPK2 inhibition, genetic ablation of flagellar motility sensitises them to loss of MPK2 activity. Thus, flagellar motility acts as a mechanical buffer during promastigote division, explaining why non-motile amastigotes are vulnerable to MPK2 inhibition. Together, our findings provide a systems level framework for Leishmania signalling and demonstrate that cellular architecture and biomechanics dictate protein kinase dependency across developmental stages of the parasite.

cell biology↗
bioRxiv2026

Single-cell Transcriptome-Proteome-PTMome(x8)-Metabolome (scTPPM) co-profiling decodes multimodal regulation during mouse oocyte meiosis

Single-cell multi-omics technology is crucial for comprehensive characterization of cellular molecular features. Despite recent advances in single-cell multi-omics approaches, co-profiling of nucleic acids, proteins, metabolites, and post-translational modifications (PTMs) within the same cell individuals remains major challenges. Here, we developed a single-cell transcriptome, proteome, PTMome, and metabolome (scTPPM) profiling approach, enabling simultaneous analysis of transcriptomes, proteomes, eight types of PTMs (e.g., phosphorylation, methylation, ubiquitination, O_GlcNAc, acetylation, crotonylation, lactylation, and succinylation), and metabolomes in individual cells. We demonstrated the scTPPM's performance in tumor cell lines and oocyte samples, reaching total identification and quantification of 27,668 genes, 4,847 proteins, 1,508 PTM sites, and 680 metabolites in single mouse oocytes. We profiled previously-uncharacterized dynamic crosstalk among the entire metabolic pathways from metabolic enzyme expression and regulation to metabolite-mediated post-translational modifications during oocyte maturation. In summary, scTPPM provided an effective workflow for multi-omics co-profiling in single cells, enabling multi-layered insights into complex regulatory network.

cell biology↗
bioRxiv2026

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗