ABSTRACT
The serotonin 2A receptor (5-HT2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT2A R has marked distinction with ligand-bound conformations of 5-HT2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier.
