Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
Hormones and neurotransmitters transduce signals through G protein-coupled receptors (GPCR). Despite their common signaling pathways, however, the responses they elicit have different temporal patterns. To reveal the molecular basis for these differences we have developed a generally applicable fluorescence-based technique for real-time monitoring of the activation switch of GPCRs in living cells. We used such direct measurements to investigate the activation of the alpha(2A)-adrenergic receptor (alpha(2A)AR; neurotransmitter) and the parathyroid hormone receptor (PTHR; hormone) and observed much faster kinetics than expected: approximately 40 ms for the alpha(2A)AR and approximately 1 s for the PTHR. The different switch times are in agreement with the different receptors' biological functions. Agonists and antagonists could rapidly switch the receptors on or off, whereas a partial agonist caused only a partial signal. This approach allows the comparison of agonist and partial agonist intrinsic activities at the receptor level and provides evidence for millisecond activation times of GPCRs.
Vilardaga
Jean-Pierre
JP
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, D-97078 Würzburg, Germany.
Bünemann
Moritz
M
Krasel
Cornelius
C
Castro
Mariàn
M
Lohse
Martin J
MJ
D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D003000:Clonidine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D011485:Protein Binding; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013050:Spectrometry, Fluorescence / Q000379:methods*
C547768:ADRA2A protein, human; D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins; D003000:Clonidine