Abstract
G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. Activated GPCRs can undergo agonist-induced phosphorylation by G protein receptor kinases (GRKs) and second messenger-dependent protein kinases such as protein kinase A (PKA). Here, we characterize spatially segregated subpopulations of β2-adrenergic receptor (β2AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. GRKs primarily label monomeric β2ARs that undergo endocytosis, whereas PKA modifies dimeric β2ARs that remain at the cell surface. In hippocampal neurons, PKA-phosphorylated β2ARs are enriched in dendrites, whereas GRK-phosphorylated β2ARs accumulate in soma, being excluded from dendrites in a neuron maturation-dependent manner. Moreover, we show that PKA-phosphorylated β2ARs are necessary to augment the activity of L-type calcium channel. Collectively, these findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell.
Highlights
G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states
Using super-resolution structured illumination microscopy (SIM), we found that after acute stimulation with the βAR agonist isoproterenol (ISO, 30 s or 1 min), both protein kinase A (PKA)- and GRKphosphorylated β2-adrenergic receptor (β2AR) are primarily segregated at the plasma membrane (PM) of HEK293 cells (Fig. 1b, top panel; Fig. 1c, d, Pearson’s coefficient 0.078 ± 0.016 for ISO 30 s and 0.058 ± 0.015 for ISO 1 min, mean ± s.e.m, three independent experiments)
This study reveals that a GPCR (β2AR) can be present in functionally distinct subpopulations that are distributed at different subcellular locations in a single cell
Summary
G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. We characterize spatially segregated subpopulations of β2-adrenergic receptor (β2AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. We show that PKA-phosphorylated β2ARs are necessary to augment the activity of L-type calcium channel These findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell. Our results show that GRKs and PKA selectively label two distinct subpopulations of β2AR that are spatially segregated on the plasma membrane and undergo distinct membrane trafficking in both fibroblasts and neurons. These two subpopulations exert distinct functions in modulating L-type calcium channel (LTCC) activity and neuron excitability
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