Fluorescence studies of homooligomerization of adenosine A(2A) and serotonin 5-HT(1A) receptors reveal the specificity of receptor interactions in the plasma membrane.
Journal Title: Pharmacological Reports - Year 2007, Vol 59, Issue 4
Abstract
The concept that G protein-coupled receptors (GPCRs) function as oligomers has been widely accepted, however, different methodologies often used to study the phenomenon of GPCR interactions do not allow, as yet, for any generalization as to whether di- or oligomers are formed constitutively or are ligand-promoted. Here, we report on the use of three independent biophysical approaches based on the Förster resonance energy transfer to study the adenosine A(2A) and serotonin 5-HT(1A) receptor (tagged with derivatives of green fluorescence protein, CFP - fluorescence donor and YFP - fluorescence acceptor) homodimerization in the plasma membrane of transiently transfected HEK 293 cell line. Homodimers of A(2A) and 5-HT(1A) receptors are formed constitutively, however, specific ligands regulate the degree of these interactions: agonists (CGS 21680 and 8-OH-DPAT, respectively) further enhanced while antagonists (SCH 58216 and methysergide) diminished the dimer formation. Although the acceptor photobleaching with the use of confocal microscopy as well as the fluorescence lifetime microscopy gave similar results, we strongly recommend the latter technique as a highly sensitive and quantitative approach to that kind of the study. The additional proof of specificity of the observed results is provided by the studies of interaction of adenosine A(2A) and serotonin 5-HT(1A) receptors with the alpha subunits of G proteins. The A(2A) receptor interacted with Galphas and 5-HT(1A) receptor - with Galphai, while physical interaction of these receptors with no appropriate alpha subunits partners (A(2A)-Galphai and 5HT(1A)-Galphas) has not been observed, despite the identical level of overexpression of proteins in all studied combinations.
Authors and Affiliations
Sylwia Łukasiewicz, Ewa Błasiak, Agata Faron-Górecka, Agnieszka Polit, Magdalena Tworzydło, Andrzej Górecki, Zygmunt Wasylewski, Marta Dziedzicka-Wasylewska
Keywords
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