Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

doi:10.1073/pnas.0704257104

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

*Département de Biochimie and
^‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7; and
^†Biozentrum, University of Basel, CH-4056 Basel, Switzerland

Edited by Anthony Pawson, University of Toronto, Toronto, Canada, and approved September 11, 2007 (received for review May 7, 2007)

Abstract

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Gα_s protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive β-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

Footnotes

^§To whom correspondence should be addressed. E-mail: stephen.michnick{at}umontreal.ca
Author contributions: E.S. and S.W.M. designed research; E.S., S.A., N.B., C.R.L., and B.N. performed research; E.S. and S.W.M. analyzed data; and E.S., M.B., and S.W.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0704257104/DC1.
Abbreviations:

GPCR,

G protein-coupled receptor;

PCA,

protein-fragment complementation assay;

PKA,

protein kinase A;

Rluc,

Renilla luciferase;

PDE,

phosphodiesterase;

β2AR,

β-2 adrenergic receptor;

V2R,

vasopressin-2 receptor;

AVP,

arginine–vasopressin.