Browsing by Subject "G protein-coupled receptor"

G protein-coupled receptors (GPCRs), a class of critical regulators of mammalian physiology, can initiate unique functional responses depending on the subcellular compartment of their activation. Yet, how endosomal receptors transduce location-biased outcomes remains poorly understood. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have largely focused on the biochemical aspect of this regulation through dissection of the relevant factors. Here, we assessed the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism coordinating the transduction of spatially-biased responses. We focused on the beta2-adrenergic receptor (β2AR), a prototypical GPCR that signals from early endosomes via cyclic AMP (cAMP) production. We examined the role of endosome positioning in the context of gene transcription as a representative signaling readout, because endosomal β2ARs are well-known to selectively stimulate transcriptional reprogramming. First, we developed subcellular-localized cAMP and protein kinase A (PKA) sensors to enable selective quantification of endosomal GPCR-mediated activity. We also generated two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cAMP-dependent transcription with single-cell resolution. We next overcame a technical challenge that has hindered the direct assessment of the functional role of endosome positioning by devising a strategy to selectively and rapidly redistribute receptor-containing endosomes ‘on command’ in intact cells without perturbing their biochemical composition. By combining these readouts with rapid endosome relocalization, we established that disruption of native endosome positioning inhibits the initiation of the endosome-dependent responses. Lastly, utilizing the cAMP and PKA sensors, we demonstrated a prominent mechanistic role of local PKA activity and phosphodiesterase (PDE)-mediated cAMP hydrolysis in this process. This dissertation work, therefore, illuminates a novel mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially-selective receptor signaling.

G protein-coupled receptors (GPCRs) represent the largest and the most versatile family of cell surface receptors. Members of this receptor family translate diverse extracellular cues to intracellular responses, and are commonly targeted for medicinal therapeutics. In the current model of GPCR signaling, agonist binging not only initiates G protein-mediated signaling through generation of second messengers such as cyclic AMP and diacylglycerol, but also through the multifunctional adaptor protein beta-arrestin acting as a signal transducer. While some ligands have balanced agonist activity defined as equal efficacy for G protein and beta-arrestin-mediated pathways, other ligands stimulates GPCR signaling preferentially through beta-arrestin, a concept know as beta-arrestin-biased agonism.

The beta1 and beta2 adrenergic receptors (betaARs) are the predominant GPCR subtypes expressed in the heart, and play an important role in the pathophysiology of human heart disease. Of the four families of G alpha-proteins (G alpha s, G alpha i/o, G alpha q/11 and G alpha 12/13), beta1ARs are recognized as classical G alpha s-coupled receptors since agonist binding promotes coupling to heterotrimeric G protein (G alpha-beta-gamma) triggering dissociation of G alpha s from G beta-gamma. Here, we identify a new signaling mechanism unlike that previously for any known G alpha s-coupled receptor whereby the inhibitory G protein (G alpha i) is required for beta1AR-mediated beta-arrestin-biased signaling. Stimulation with the beta-arrestin-biased agonist carvedilol induces switching of the beta1AR from a classical G alpha s-coupled receptor to a G alpha i-coupled receptor and stabilizes a unique receptor conformation required for beta-arrestin-mediated signaling. Recruitment of G alpha i was not induced by any other betaAR agonist or antagonist screened, nor was it required for beta-arrestin-bias activated by the beta2AR subtype of the betaAR family.

We also found that G alpha i is involved in the membrane stretch-induced signaling activated by the angiotensin II type 1 receptor (AT1R), another GPCR mediating a variety of physiological responses and is commonly targeted for cardiac drug therapy. It is appreciated that the AT1R can function as a mechanical sensor. When activated by mechanical stretch, AT1Rs transmit signaling through beta-arrestin, rather than the G protein pathways. To date, the ligand-independent membrane stretch-induced AT1R conformation that triggers signaling is thought to be the same as that induced by a beta-arrestin-biased agonist, which can selectively engage beta-arrestin and prevent G protein coupling. Here we show that membrane stretch promotes a distinct biased conformation of the AT1R that couples to G alpha i. In contrast, recruitment of G alpha i was not induced by the balanced agonist angiotensin II, nor by the beta-arrestin-biased agonist TRV120023. The stretch-triggered G alpha i coupling induces the recruitment and a unique conformational change in beta-arrestin2 allowing for downstream signaling such as EGFR internalization and ERK phosphorylation.

Taken together, we identified a previously unrecognized role for G alpha i in beta-arrestin-biased GPCR signaling, and suggests that the concept of beta-arrestin-bias may need to be refined to incorporate the selective bias of receptors towards distinct G protein subtypes. We also demonstrate that different mechanisms for beta-arrestin bias may be operative between the signaling induced by distinct receptor activation modes, in the case of AT1R, the beta-arrestin-biased agonists and by the ligand-independent mechanical stretch.

These data underscore the complexity of beta-arrestin-biased agonism and have important implications when considering the development of new therapeutic ligands to selectively target beta-arrestin-biased signaling pathways.