Campbell Krusemark

G protein coupled receptors, or GPCRs, are the largest family of membrane receptors in humans and can be found throughout the body in numerous cell types. Crucial to intracellular signaling, GPCRs can induce their effects through various pathways depending on the composition of the associated G protein. However, these receptors also have the ability to recruit additional effector proteins in order to modify or terminate their signal. Specifically, subsequent recruitment of a G protein receptor kinase and an arrestin protein will cause the receptor to arrest its signaling via endocytosis. Recently, it has been suggested that the intracellular trafficking of the receptor can alter receptor signaling in addition to halting the signal altogether. As the receptor is localized in intracellular compartments, recent studies suggest that they can continue to signal with unique physiological effects than the same receptor on the outer membrane. Thus, receptor trafficking may be an important determinant when characterizing mechanisms of drug action. Despite this, there are few investigative methods to understand these pathways. In this study we offer bystander bioluminescence resonance energy transfer (BRET) as a method to quantitatively elucidate the trafficking of the neurotensin receptor 1(NTSR1), a GPCR. Using the Renilla-derived chromophore luciferase (Rluc) and the yellow fluorescent protein Venus, spectrometric BRET signals were derived from NTSR1 internalization following activation with the endogenous ligand, neurotensin, and SBI-553, a β-arrestin biased allosteric modulator of the receptor. Analyzing these signals can reveal the kinetics and dynamics of receptor trafficking and provide insight into future localization studies.