Membrane organization of the human serotonin(1A) receptor monitored by detergent insolubility using GFP fluorescence.
Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT(1A) receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin(1A) receptor tagged to enhanced yellow fluorescent protein (5-HT(1A)R-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT(1A) receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT(1A)R-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT(1A)R-EYFP provide new insights into the membrane organization of the 5-HT(1A) receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.
Kalipatnapu
Shanti
S
Centre for Cellular and Molecular Biology, Hyderabad, India.
Chattopadhyay
Amitabha
A
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003902:Detergents / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000737:chemistry*; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry* / Q000235:genetics
D003902:Detergents; D044282:Receptor, Serotonin, 5-HT1A; D049452:Green Fluorescent Proteins; D017830:Octoxynol