Compartmentalized GPCR Signaling from Intracellular Membranes

Abstract

G protein-coupled receptors (GPCRs) are integral membrane proteins that transduce a wide array of inputs including light, ions, hormones, and neurotransmitters into intracellular signaling responses which underlie complex processes ranging from vision to learning and memory. Although traditionally thought to signal primarily from the cell surface, GPCRs are increasingly being recognized as capable of signaling from intracellular membrane compartments, including endosomes, the Golgi apparatus, and nuclear membranes. Remarkably, GPCR signaling from these membranes produces functional effects that are distinct from signaling from the plasma membrane, even though often the same G protein effectors and second messengers are activated. In this review, we will discuss the emerging idea of a "spatial bias" in signaling. We will present the evidence for GPCR signaling through G protein effectors from intracellular membranes, and the ways in which this signaling differs from canonical plasma membrane signaling with important implications for physiology and pharmacology. We also highlight the potential mechanisms underlying spatial bias of GPCR signaling, including how intracellular membranes and their associated lipids and proteins affect GPCR activity and signaling.

Keywords: Endosomes; GPCR; Golgi; Nuclear membrane; Signaling; Trafficking.

Fig. 1.. Examples of localization and signaling of GPCRs from intracellular membranes.

In addition to the plasma membrane GPCRs localize and signal through G proteins in compartments along the biosynthetic pathway (solid arrows) and the endolysosomal pathway (dashed arrows). Non-canonical GPCR signaling through G proteins in these membranes can proceed by distinct interactions, such as a PTHR-G protein-arrestin signaling complex, and produce distinct downstream signaling effects, including but not limited to sustained signaling, transcriptional responses, and effects on cell growth, neurotransmission, and the perception of pain.

Fig. 2:. Tools for studying GPCR spatial signaling bias.

a. Conformational biosensors including nanobodies and miniG proteins recognize active GPCR or Gs conformations. When expressed in cells these sensors will translocate from the cytosol to membranes containing active GPCR or G protein. If tagged with a fluorescent protein, translocation of the sensor can be visualized by confocal microscopy. Inset: HEK cell expressing Flag-DOR and venus-miniGsi and treated with 10μM SNC80 agonist. Accumulation of the miniG sensor is visible on endosomal membranes (yellow arrow), scale bar=1.5μm. b. Cholestanol-conjugated ligands incorporate into the lipid bilayer, are internalized, and accumulate in endosomes. When concentrated in endosomes, these ligands can provide prolonged and specific agonism or antagonism of endosomal GPCRs. Whether these endosomally-targeted antagonists also inhibit plasma membrane GPCRs over prolonged time periods, and if so, to what levels is not clear. c. To isolate GPCR signaling from intracellular sites, cells can be treated with a membrane permeable agonist along with a membrane impermeable antagonist. The permeable agonist can access and activate intracellular GPCRs, while the impermeable antagonist acts only on GPCRs in the plasma membrane, preventing agonist from binding to these sites. d. Fluorescent FRET sensors for PKA phosphorylation events or second messengers like cAMP produced downstream of GPCR activation can be targeted to specific cellular membranes. Localization of these sensors to specific membranes provides a readout of local signaling events. Second messenger like cAMP or phosphorylation of a kinase motif on the sensor leads to a conformational change in the sensor. Changes in cellular levels of cAMP or kinase activity are measured as the ratio between donor fluorophore (D) and acceptor fluorophore (A) emission for a ratiometric FRET sensor, like the one depicted. e. Nanobodies which recognize the active conformation of GPCRs specifically inhibit GPCR signaling from intracellular sites. Rapamycin induces dimerization of FRB and FKBP domains fused to a nanobody or targeted to the organelle of interest, respectively. Dimerization leads to a high local concentration of nanobody which binds to active GPCRs. These nanobodies compete with endogenous G proteins for interaction with the active receptor, interfering with G protein activation and signaling in the organelle of interest. f. Caged ligands are chemically modified so that they are inactive at the target receptor until uncaging. Local uncaging of caged ligands by a specific wavelength of light leads to photolysis of the caged ligand, freeing the active ligand. The free ligand can then bind to the target receptor.