Abstract Body (Do not enter title and authors here): Traditional G Protein-Coupled Receptors (GPCR) targeted therapeutics have primarily focused on developing ligands that bind to the orthosteric site. However, receptors live in numerous distinct active and inactive conformational states. As a result, the receptor surface contains many different sites that can targeted by allosteric modulators. Allosteric modulators are attractive drug targets because they can bias signaling for already FDA-approved orthosteric therapeutics. However, a major limitation of many allosteric modulators is that it is difficult to localize these to subcellular compartments. Thus, we engineered a peptide inhibitor, Ga_q carboxy tail (GqCT), to fine-tune signaling at different subcellular locations. Classically, GqCT serves as a Ga_q inhibitor, by binding to receptor-transducer binding pocket and abolishes downstream signaling by outcompeting endogenous Ga_q,. For example, global expression of GqCT diminishes a₁AR-mediated inositol production in cardiac cells, although little is known about its function with other Ga_q coupled receptors. We hypothesized that subcellular tagged GqCT could produce unique signaling signatures for Ga_q coupled receptors such as a₁AR, V₁R, and AT₁R.

Leveraging our novel ERK and RhoA BRET-based biosensors we found that subcellular GqCT diminished signaling for a₁AR and AT₁R, whereas for V₁R there were little changes in ERK signaling. This data suggests that GqCT may serve distinct functions for vasoactive GPCRs. We next evaluated β-arrestin recruitment and found that plasma membrane-localized GqCT (PM-GqCT) increased recruitment for both the AT₁R and V₁R. Next, we evaluated the internalization of AT₁R and found that numerous locations such as PM-GqCT and Actin-GqCT enhanced internalization, whereas endosomal-GqCT did alter internalization compared to control. This data suggests that GqCT is a β-arrestin biased allosteric modulator that serves to enhance internalization for the AT₁R.

In summary, we repurposed the Ga_q inhibitor, GqCT, by tagging it to different subcellular and found it inhibits signaling a₁AR and AT₁R, and conversely enhanced ERK signaling for V₁R. This could be contributed, in part, by GqCT serving as an β-arrestin biased allosteric modulator. Further work will evaluate the function of GqCT in physiologically relevant cells such as vascular smooth muscle and cardiac cells. This work elucidates the unique nature of subcellular-GqCT on signaling and internalization.