Fluorescence studies of homooligomerization of adenosine A2A and serotonin 5-HT1A receptors reveal the specificity of receptor interactions in the plasma membrane.
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 A2A and serotonin 5-HT1A 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 A2A and 5-HT1A 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 A2A and serotonin 5-HT1A receptors with the alpha subunits of G proteins. The A2A receptor interacted with G alpha s and 5-HT1A receptor - with G alpha i, while physical interaction of these receptors with no appropriate alpha subunits partners (A2A-G alpha i and 5HT1A-G alpha s) has not been observed, despite the identical level of overexpression of proteins in all studied combinations.
Łukasiewicz
Sylwia
S
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, PL 30-387 Kraków, Poland.
Błasiak
Ewa
E
Faron-Górecka
Agata
A
Polit
Agnieszka
A
Tworzydło
Magsalena
M
Górecki
Andrzej
A
Wasylewski
Zygmunt
Z
Dziedzicka-Wasylewska
Marta
M
D000465:Algorithms; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D008164:Luminescent Proteins; D043705:Receptor, Adenosine A2A; D044282:Receptor, Serotonin, 5-HT1A