Analysis of receptor oligomerization by FRAP microscopy.
Here we describe an approach to investigate di- or oligomerization of transmembrane receptors in living cells with fluorescence recovery after photobleaching (FRAP). We immobilized a defined fraction of receptors with antibodies and then measured lateral mobility of the nonimmobilized fraction by FRAP. We validated this approach with CD86 and CD28 as monomeric and dimeric reference proteins, respectively. Di- or oligomerization of G protein-coupled receptors is strongly debated. We studied human beta-adrenergic receptors as prototypical G protein-coupled receptors and found that beta(1)-AR shows transient interactions whereas beta(2)-AR can form stable oligomers. We propose that this FRAP method can be widely applied to study di- or oligomerization of cell-surface proteins.
Dorsch
Sandra
S
University of Würzburg, Institute of Pharmacology and Toxicology, Versbacher Strasse 9, Würzburg, Germany.
Klotz
Karl-Norbert
KN
Engelhardt
Stefan
S
Lohse
Martin J
MJ
Bünemann
Moritz
M
D030301:Activin Receptors, Type II / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D051940:B7-2 Antigen / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D018106:CD28 Antigens / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D032383:Myocytes, Cardiac / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D051940:B7-2 Antigen; D001426:Bacterial Proteins; D018106:CD28 Antigens; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D030301:Activin Receptors, Type II; C425584:activin receptor type II-B