G-protein-dependent cell surface dynamics of the human serotonin1A receptor tagged to yellow fluorescent protein.
Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive, behavioral, and developmental processes. The serotonin(1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors and is the most extensively studied among the serotonin receptors. Several aspects of serotonin(1A) receptor biology such as cellular distribution and signal transduction characteristics are technically difficult to address in living cells on account of the inability to optically track these receptors with fluorescence-based techniques. We describe here the characterization of the serotonin(1A) receptor tagged to the enhanced yellow fluorescent protein (EYFP) stably expressed in Chinese hamster ovary (CHO) cells. These receptors were found to be essentially similar to the native receptor in pharmacological assays and can therefore be used to reliably explore aspects of receptor biology such as cellular distribution and dynamics on account of their intrinsic fluorescent properties. Analysis of the cell surface dynamics of these receptors by fluorescence recovery after photobleaching (FRAP) experiments has provided novel insight into the molecular mechanism of signal transduction of serotonin(1A) receptors in living cells. Interestingly, addition of pharmacologically well-characterized ligands or activators of G-proteins altered the diffusion characteristics of the receptor in a manner consistent with the G-protein activation model. These results demonstrate, for the first time, that membrane dynamics of this receptor is modulated in a G-protein-dependent manner.
Pucadyil
Thomas J
TJ
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Kalipatnapu
Shanti
S
Harikumar
Kaleeckal G
KG
Rangaraj
Nandini
N
Karnik
Sadashiva S
SS
Chattopadhyay
Amitabha
A
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000494:pharmacology; D017607:Aluminum Compounds / Q000494:pharmacology; D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D036681:Fluorescence Recovery After Photobleaching; D005459:Fluorides / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D010879:Piperazines / Q000494:pharmacology; D011869:Radioligand Assay; D044282:Receptor, Serotonin, 5-HT1A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017607:Aluminum Compounds; D000631:Aminopyridines; D001426:Bacterial Proteins; D008024:Ligands; D008164:Luminescent Proteins; D010879:Piperazines; C054562:yellow fluorescent protein, Bacteria; D044282:Receptor, Serotonin, 5-HT1A; C050992:tetrafluoroaluminate; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C099463:4-(2' methoxyphenyl)-1-(2'-(N-(2''-pyridinyl)-4-fluorobenzamido)ethyl)piperazine; D020962:Heterotrimeric GTP-Binding Proteins; D005459:Fluorides