Mapping distinct vasodilator-induced phosphorylation patterns in human vascular smooth muscle: A quantitative phosphoproteomic approach
Authors: Sloniecka Marta , Harris Joscelyn , Eyers Claire , Dart Caroline 
Published: May 2023

In vascular smooth muscle cells (VSMCs), the diffusive second messenger, 3'-5'-cyclic adenosine monophosphate (cAMP), relays crucial blood vessel-widening 'relaxation' signals from surface receptors to downstream effectors. These in turn modulate multiple proteins to relax VSMCs and increase arterial blood flow. Different cAMP-coupled receptors are believed to modulate distinct populations of proteins, leading to variation in the speed, degree and longevity of relaxation in response to different vasodilators, although this has never been fully investigated. In humans, genetic variations in proteins that control cAMP production and degradation are associated with variability in vascular reactivity and susceptibility to hypertension, but mechanistically we know surprisingly little about cAMP signalling pathways in VSMCs. Here we use quantitative phosphoproteomics to map spatial and temporal protein phosphorylation patterns in human coronary smooth muscle cells (HCASMC) in response to different vasodilators. Primary HCASMC were stimulated with either adenosine (10μM), isoprenaline (1μM), calcitonin gene-related peptide (CGRP; 10nM) or epoprostenol (10nM) for 2 mins, 10 mins or 1 hour. Quantitative phosphoproteomics exploiting Tandem Mass Tag (TMT)-based isobaric labelling was used to correlate, in a high-throughput unbiased manner, changes in phosphorylation of all cellular proteins. Four biological replicates were analyzed. Functional enrichment analysis was performed with the DAVID bioinformatics database. 721 phosphopeptides with at least one site of phosphorylation were identified (all time points). 329 of these peptides had a significant p-value (p<0.05) when compared to untreated cells. 90 p-value significant phosphopeptides were identified for adenosine, 72 for isoprenaline, 68 for CGRP and 70 for epoprostenol, with typically 20-30 phosphopeptides shared between different vasodilators. Consistent with vasodilatory function, all agonists phosphorylated proteins associated with actin binding activity at early time points, with a general shift to proteins involved in RNA binding following prolonged exposure. Distinct spatio-temporal phosphorylation patterns were also evident for different vasodilators: For epoprostenol, phosphorylation activity peaked in the nucleus at 2 mins before declining, while nuclear activity induced by all other vasodilators increased over time. Isoprenaline-induced phosphorylation appeared confined to the plasma membrane, cytoskeleton and cytoplasmic regions at all time points, whereas adenosine, CGRP and epoprostanol induced additional activity in the Golgi apparatus, endoplasmic reticulum and mitochondria at 2-10 mins.Our results demonstrate vasodilator-specific signalling compartmentation in HCASMCs, likely controlled by the ‘funnelling’ of cAMP to discrete, localised effectors. Future work should define the role of specific phosphodiesterases in shaping this signalling and determine whether these patterns are Supported by BBSRC Award BB/V002767/1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.