br Conclusion br Conflict of interest

Conclusion
Conflict of interest
Acknowledgment The authors acknowledge the grant support of the Tehran University of Medical Sciences (94-02-49-29647).
Introduction Earlier in vitro observations have also established that the human 5-HT2A, while very similar to the rat isoform, exhibits considerably different behaviors (Bhattacharya et al., 2010). For example, the rat isoform of the 5-HT2A, the S291 residue shows hampered internalization (Raote et al., 2013). Therefore, the interactions of the S291A mutant of the human 5-HT2A with dopamine were also investigated.
Experimental Procedures
Results
Discussion Our results highlight the fact that different residues of the silibinin play a role in ligand-dependent downstream effects which would be related to its interactions with signaling partners. It also underscores the idea of functional selectivity, where different ligands bring about different signaling effects via the same receptor. In this study, we explored some aspects of this phenomenon in the action of dopamine, a novel, endogenous ligand on 5-HT2A, whose distribution greatly overlaps with that of 5-HT in the CNS. Our observations lead us to conclude that functional selectivity shown by 5-HT2A involves multiple residues and that the residues that we have interrogated, being on the cytoplasmic segments of the receptor, are responsible for differences “in both, proximal as well as distal events” during receptor signaling (Raote et al., 2013). As far as we are aware, this paper is the first to demonstrate that DA acts as a weak efficacy or partial agonist at the human isoform of 5-HT2A and has identified some of the residues that are critical to the signaling process. To our knowledge, this is the first report where the residues of a neurotransmitter receptor (5-HT2A) have been examined for their roles in its functional selectivity with respect to the activity of another non-canonical, endogenous neurotransmitter (DA). It is also important to note that DA behaves similar to the other ligands tested, with respect to the phenotypes that have been investigated in this study, albeit with lower efficacy. Of these, intracellular calcium accumulation and receptor internalization, which are stereotypic responses for this receptor, can be classified as immediate responses (second messenger), whereas formation of stress fibers, a novel phenotype, can be considered a phenotype that is further downstream of the signaling pathways that are activated. However, the involvement of different residues of the receptor for signaling via different ligands, specifically DA, suggests ‘functional selectivity’ of 5-HT2A; either at the level of conformational change when activated, or with altered interaction with signaling partners after ligand binding. It is evident that the ability to bring about internalization of the receptor, or activating the PLC-IP3-Ca2+ pathway or even the formation of stress fibers is hampered among the various mutants, which could be a result of compromised ability to interact with downstream signaling partners. The T307 and S291 residues are both located in the third intracellular loop. This region of the receptor has previously been shown to play an important role in “effector coupling specificity” (Oksenberg et al., 1995). Our data suggest that one of these, T307, is necessary specifically for internalization of the receptor by DA. Although the rodent isoforms of the receptor are the more popular models of study, owing to the advantage of being able to extend in vitro experiments to in vivo behavior, it has been reported that the human isoform exhibits differences from the rat isoform (Bhattacharya et al., 2010). In keeping with these observations, our study also arrives at a similar conclusion that some residues (S291), although important for trafficking of the rat isoform of the 5-HT2A, do not seem to play a role in the human isoform of the receptor. This further highlights the need to study receptor isoforms individually and has obvious clinical implications (Raote et al., 2013).