Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network

Abstract

GPCR-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale whereas endosomes typically localize microns from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunit to direct downstream signaling.

Location bias contributes to functionally selective responses of biased CXCR3 agonists

“…CXCL10 and CXCL11 both demonstrated a ~40% decrease in cAMP inhibition when receptor internalization was inhibited, even though the chemokines were able to promote different amounts of total receptor internalization (Figure 3D-3I). cAMP gradients can exist in micro or nanodomains within the cell, and endosomal cAMP production can be critical for nuclear translocation of effectors like PKA (Calebiro and Maiellaro, 2014;Musheshe et al, 2018;Peng et al, 2021). Using an EPAC 10 subcellular locations (Figure 4F-4G and 4I-4J).…”

Endosomal cAMP production broadly impacts the cellular phosphoproteome

“…In contrast to the V2R, cAMP signaling from the β2-AR is transient, and the effect of endosomal activation on global cAMP production is small (Fig. S3c) 5,21,22 . Yet, intracellular β2-ARs were shown to be essential for eliciting downstream transcriptional responses 5,22,23 .…”

“…S3c) 5,21,22 . Yet, intracellular β2-ARs were shown to be essential for eliciting downstream transcriptional responses 5,22,23 . Similarly to what we observed with overexpressed V2R, β2-AR stimulation led to increase in PKA substrate phosphorylation, which required receptor internalization (Fig.…”

The OCT2 Transporter Regulates Dopamine D1 Receptor Signaling at the Golgi Apparatus

“…Binding of cAMP to the PKA regulatory subunit induces rapid dissociation and activation of the PKA catalytic subunit (Figure 5a) (68,69). To test whether cAMP generation by Golgi-localized D1DR/Gs complex results in the activation of PKA at the perinuclear/Golgi, we utilized a previously described HEK293T knock-in cell line expressing a split fluorescent protein, labeling native PKA catalytic subunit gene with GFP (PKAcat-GFP) (35,70). Stimulation of HEK293T PKAcat-GFP knock in cell lines expressing D1DR with 10nM SKF81297, a concentration that activates both pools of D1DR (Supplementary Figure 3), resulted in rapid dissociation of PKAcat-GFP from the perinuclear/Golgi membranes (Figure 5b top panel).…”

“…However, evidence from the past decade suggests that for some GPCRs endocytosis might in fact activate a second phase of acute or prolonged Gas-mediated cAMP response from the endosomes (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Recent studies further support this notion by providing evidence that cAMP generation by activated receptors at the endosome are necessary in regulating transcriptional responses that are distinct from those elicited by activation of the plasma membrane receptor pool (30)(31)(32)(33)(34)(35).…”