No abstract

β-Adrenergic receptors (βAR) and D2-like dopamine receptors (which include D2-, D3- and D4-dopamine receptors) activate Gs and Gi, the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). β2-Adrenergic receptors (β2AR) and D4-dopamine receptors (D4DR) co-immunoprecipitated when co-expressed in HEK 293 cells, suggesting the existence of a signalling 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, β2AR, D4DR, G protein subunits (Gαi1 and the Gβ1γ2 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 β2AR, D4DR, G proteins and AC were closely associated in a functional signalling complex in cellulo. Furthermore, BRET experiments indicated that although activation of Gi caused a conformational change within the heterotrimeric protein, it did not cause the Gβγ heterodimer to dissociate from the Gαi1 subunit. Evidence for the presence of a signalling 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 D2-like dopamine receptors and AC.