D 2-like dopamine and β-adrenergic receptors form a signaling complex that integrates G s- and G i-mediated regulation of adenylyl cyclase

R. Victor Rebois, Karl Maki, Julie A. Meeks, Peter H. Fishman, Terence E. Hébert, John K. Northup

Research output: Contribution to journalArticlepeer-review

Abstract

β-Adrenergic receptors (βAR) and D 2-like dopamine receptors (which include D 2-, D 3- and D 4-dopamine receptors) activate G s and G i, the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). β 2-Adrenergic receptors (β 2AR) and D 4-dopamine receptors (D 4DR) co-immunoprecipitated when co-expressed in HEK 293 cells, suggesting the existence of a signaling complex containing both receptors. In order to determine if these receptors are closely associated with each other, and with other components involved in G protein-mediated signal transduction, β 2AR, D 4DR, G protein subunits (Gα i1 and the Gβ 1γ 2 heterodimer) and AC were tagged so that bioluminescence resonance energy transfer (BRET) could be used to monitor their interactions. All of the tagged proteins retained biological function. For the first time, FlAsH-labeled proteins were used in BRET experiments as fluorescent acceptors for the energy transferred from Renilla luciferase-tagged donor proteins. Our experiments revealed that β 2AR, D 4DR, G proteins and AC were closely associated in a functional signaling complex in cellulo. Furthermore, BRET experiments indicated that although activation of G i caused a conformational change within the heterotrimeric protein, it did not cause the Gβγ heterodimer to dissociate from the Gα i1 subunit. Evidence for the presence of a signaling complex in vivo was obtained by purifying βAR from detergent extracts of mouse brain with alprenolol-Sepharose and showing that the precipitate also contained both D 2-like dopamine receptors and AC.

Original languageEnglish (US)
Pages (from-to)2051-2060
Number of pages10
JournalCellular Signalling
Volume24
Issue number11
DOIs
StatePublished - Nov 1 2012
Externally publishedYes

Keywords

  • Adenylyl cyclase
  • Alprenolol-Sepharose
  • Bioluminescence resonance energy transfer
  • D -like dopamine receptors
  • Heterotrimeric G proteins
  • Signaling complexes
  • β-Adrenergic receptors

ASJC Scopus subject areas

  • Cell Biology

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