title: Investigating signaling consequences of GPCR trafficking in the endocytic pathway.
abstract: Ligand-dependent regulation of adenylyl cyclase by the large family of seven-transmembrane G protein-coupled receptors (GPCRs) represents a deeply conserved and widely deployed cellular signaling mechanism. Studies of adenylyl cyclase regulation by catecholamine receptors have led to a remarkably detailed understanding of the basic biochemistry of G protein-linked signal transduction and have elaborated numerous mechanisms of regulation. Endocytosis of GPCRs plays a significant role in controlling longer-term cellular responses, such as under conditions of prolonged or repeated receptor activation occurring over a course of hours or more. It has been more challenging to investigate regulatory effects occurring over shorter time intervals, within the minutes to tens of minutes spanning the time course of many acute cyclic AMP (cAMP)-mediated signaling processes. A main reason for this is that biochemical methods used traditionally to assay changes in cytoplasmic cAMP concentration are limited in spatiotemporal resolution and typically require perturbing cellular structure and/or function for implementation. Recent developments in engineering genetically encoded cAMP biosensors linked to optical readouts, which can be expressed in cells or tissues and detected without cellular disruption or major functional perturbation, represent a significant step toward overcoming these limitations. Here, we describe the application of two such cAMP biosensors, one based on enzyme complementation and luminescence detection and another using Förster resonance energy transfer and fluorescence detection. We focus on applying these approaches to investigate cAMP signaling by catecholamine receptors and then on combining these analytical approaches with manipulations of receptor endocytic trafficking.
authors.affiliation: Department of Psychiatry, University of California School of Medicine, San Francisco, California, USA; Department of Cellular & Molecular Pharmacology, University of California School of Medicine, San Francisco, California, USA.
authors.forename: Roshanak
authors.initials: R
authors.lastname: Irannejad
authors.affiliation: Department of Psychiatry, University of California School of Medicine, San Francisco, California, USA; Department of Cellular & Molecular Pharmacology, University of California School of Medicine, San Francisco, California, USA.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Kotowski
authors.affiliation: Department of Psychiatry, University of California School of Medicine, San Francisco, California, USA; Department of Cellular & Molecular Pharmacology, University of California School of Medicine, San Francisco, California, USA. Electronic address: mark.vonzastrow@ucsf.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: von Zastrow
mesh_terms: D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015290:Second Messenger Systems
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: Clathrin; Cyclic AMP; Dynamin; Endosome; Fluorescence microscopy; Luminescence imaging