Functional reconstitution of the human serotonin receptor 5-HT(6) using synthetic transmembrane peptides.
Seven transmembrane (7TM) synthetic peptides mimicking the alpha-helical TM domains of the human serotonin receptor subtype-6 (5-HT(6)) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 microM. These results show that the seven individual TM domains of 5-HT(6) can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.
Lee
Won-Kyu
WK
Department of Chemistry, College of Natural Science, Kookmin University, Seoul 136-702, Republic of Korea.
Han
Jason J
JJ
Jin
Bong-Suk
BS
Boo
Doo Wan
DW
Yu
Yeon Gyu
YG
D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D008823:Micelles; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000187:drug effects; D017366:Serotonin Receptor Agonists / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D008081:Liposomes; D008823:Micelles; D010455:Peptides; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists; C097216:serotonin 6 receptor