Studying the regulation of endosomal cAMP production in GPCR signaling

Alexandre Gidon; Timothy N Feinstein; Kunhong Xiao; Jean-Pierre Vilardaga

doi:10.1016/bs.mcb.2015.10.007

Studying the regulation of endosomal cAMP production in GPCR signaling

Methods Cell Biol. 2016:132:109-26. doi: 10.1016/bs.mcb.2015.10.007. Epub 2015 Dec 24.

Authors

Alexandre Gidon¹, Timothy N Feinstein², Kunhong Xiao³, Jean-Pierre Vilardaga³

Affiliations

¹ Molecular Mechanisms of Mycobacterial Infection, Center for Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
² Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
³ Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Abstract

We describe methods based on live cell fluorescent microscopy and mass spectrometry to characterize the mechanism of endosomal cAMP production and its regulation using the parathyroid hormone (PTH) type 1 receptor as a prime example. These methods permit to measure rapid changes of cAMP levels in response to PTH, kinetics of endosomal ligand-receptor interaction, pH changes associated with receptor trafficking, and to identify the endosomal receptor interactome.

Keywords: Arrestin; Endosomal GPCR proteomics; Endosomal GPCR signaling; FRET; GPCR trafficking; PTH receptor; Receptor dynamics; TIRF.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Amino Acid Sequence
Cyclic AMP / biosynthesis*
Endocytosis
Endosomes / metabolism
Fluorescence Resonance Energy Transfer
HEK293 Cells
Humans
Hydrogen-Ion Concentration
Kinetics
Molecular Sequence Data
Protein Transport
Receptor, Parathyroid Hormone, Type 1 / chemistry
Receptor, Parathyroid Hormone, Type 1 / metabolism*
Second Messenger Systems*

Substances

Receptor, Parathyroid Hormone, Type 1
Cyclic AMP

Abstract

Publication types

MeSH terms

Substances

Grants and funding