TY - JOUR
T1 - D 2-like dopamine and β-adrenergic receptors form a signaling complex that integrates G s- and G i-mediated regulation of adenylyl cyclase
AU - Rebois, R. Victor
AU - Maki, Karl
AU - Meeks, Julie A.
AU - Fishman, Peter H.
AU - Hébert, Terence E.
AU - Northup, John K.
N1 - Funding Information:
We thank Maya Mamarbachi for making the recombinant plasmid coding for D 4 DR-PGCC, and Drs. Jurgen Wess and David R. Sibley for reviewing the manuscript. This research was supported by the Intramural Research Program of the National Institutes of Health , the National Institute on Deafness and Other Communication Disorders and the National Institute of Neurological Disorders and Stroke , and by grants to TEH from the Canadian Institutes for Health Research . TEH is a Chercheur National of the Fonds de la Recherche en Santé du Québec (FRSQ) .
PY - 2012/11
Y1 - 2012/11
N2 - 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.
AB - 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.
KW - Adenylyl cyclase
KW - Alprenolol-Sepharose
KW - Bioluminescence resonance energy transfer
KW - D -like dopamine receptors
KW - Heterotrimeric G proteins
KW - Signaling complexes
KW - β-Adrenergic receptors
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U2 - 10.1016/j.cellsig.2012.06.011
DO - 10.1016/j.cellsig.2012.06.011
M3 - Article
C2 - 22759790
AN - SCOPUS:84865327599
VL - 24
SP - 2051
EP - 2060
JO - Cellular Signalling
JF - Cellular Signalling
SN - 0898-6568
IS - 11
ER -