Abstract
Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). The EPIC-BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration-dependent negative DMR deflection that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Subtype-selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR-mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and –independent pathways.
Highlights
Label-free technologies for investigation of ligand–receptor signaling responses have been increasingly used to explore the effects of distinct ligands on receptor-dependent signaling and to elucidate their mechanisms of action (Peters et al 2010; Rocheville et al 2013)
As we showed that inhibition of receptor internalization enhanced G protein-dependent b-adenergic receptors (bAR) signaling (Fig. S2A– C) and direct interference of G protein-coupled receptor kinase (GRK)/b-arrestin signaling would promote a similar effect, we assessed the contribution of epidermal growth factor receptor (EGFR) signaling, a well-characterized downstream readout of G protein-independent bAR signaling (Maudsley et al 2000; Noma et al 2007; Tilley et al 2009), to the observed bAR-mediated Dynamic mass redistribution (DMR) response
Label-free detection of receptor responses to stimulation via DMR analysis has been utilized for several years, few studies have investigated the usefulness of this assay for dissecting endogenous receptor biology in primary cells
Summary
Label-free technologies for investigation of ligand–receptor signaling responses have been increasingly used to explore the effects of distinct ligands on receptor-dependent signaling and to elucidate their mechanisms of action (Peters et al 2010; Rocheville et al 2013). Application of DMR technology has largely focused on characterizing G protein-coupled receptor (GPCR) signaling responses in a variety of clonal cell lines with either overexpressed or endogenous levels of receptors (Codd et al 2011; Ferrie et al 2011; Tran et al 2012; Deng et al 2013; Morse et al 2013; Schrage et al 2013). There exists a paucity of studies reporting endogenous GPCR DMR responses in primary cells (Schroder et al 2010, 2011), which would enable investigators to better gauge the contribution of different receptor signaling pathways in response to ligand stimulation that may more closely reflect those in vivo
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