Agonist Induced and Constitutive Activity

The study of the interactions between agonists or antagonists and the human 5-HT2C receptor tends to show that selectivity of the 5-HT2C subtype would be more sensitive to antagonist compounds. Moreover the high pharmacological potency of 5-HT2C antagonists has been shown. Indeed the maximal responses to highly potent 5-HT2C agonists were observed when the fractional occupancy of receptors was less than one (Hartman and Northup 1996) which suggests a pool of "spare receptors" with an intrinsic constitutive activity potentially sufficient to confer a transformed phenotype in cells transfected with the 5-HT2C gene (Julius et al. 1989). Thus, the specific pharmacological actions of certain receptor antagonists may reflect the direct consequences of binding the unoccupied constitutively active 5-HT2C receptors, rather than indirect actions mediated by a blockade of binding of endogenous agonists. Other type-A GPCRs, such as adrenoceptors, seemingly have little or no constitutive activity but constitutive activation is conferred by mutations in the third cytoplasmic loop (Cotecchia et al. 1992; Kjelsberg et al. 1992; Samama et al. 1993; Ren et al. 1993). Unfortunately, this region remains a challenge for 3D modeling because of its high variability. Besides, if GPCR-type monoamine receptors have relatively long IL3 segment in comparison with bovine rhodopsin, their structure cannot be studied in crystal structures because it is partially truncated (P1-adrenoceptor) or replaced with lysosome T4 (p2-adreno-ceptor, A2A-adenosine receptor) in order to facilitate their crystallization.

Nevertheless, the recently published works about the signaling pathway in rhodopsin (Kong and Karplus 2007), reveal how the interaction network of coupled residues extends from the retinal-binding pocket to the cytoplasmic surface after 22 ns of MD runs. The interactions are mainly weakened or intensified while some charged residues of TM bundle (D2.50, E3.37, E3.49, R3.50, E6.31, K6.32, R8.50) but also in IL2 (R147) are involved and tend to be clustered in certain directions, instead of being randomly distributed. Compared with hydrophobic interactions, salt bridges and hydrogen bonds are most specific in direction, which could make them effective as molecular switches that respond to the distant perturbation of the ligand binding site. Inspecting the sequence alignment (Fig. 6.5) between RHO and 5-HT2C shows the conservation of most of these charged residues found except E3.37 in RHO mutated to a threonine in

5-HT2C and ADRB receptors. All other charged residues are either strictly conserved (D2.50, R3.50, R164, E6.31, R8.50) or homologous charged residues (D3.49, R6.32).

Thus the conductance of the signal toward the cytoplasmic surface could be the same as in bovine rhodospin. Moreover the starting structural event in bovine rho-dopsin is the formation of a Schiff base between isomerized retinal and Lys296 inducing a continuous coupling pathway from retinal to the NPxxY TM7 motif through N1.50, S3.38 and D2.50. Since the amino acid residue corresponding to the RHO Lys296 (K7.43) is Tyr358 in 5-HT2C, we suggest that polar interactions occurring between D3.32, S3.38 and Y7.43 could be perturbed by an intensified salt bridge between D3.32 and the protonated nitrogen of agonist ligands like previously discussed. This would disrupt hydrogen bond between D3.32 and Y7.43 whose the side chain conformation may switch downward to permit a hydrogen bond with the close D2.50 what would promote the signal pathway along the receptor toward the cytoplasmic region. Besides, D2.50 is also directly involved within the hydrogen bond network relayed by structural waters, as previously described in the section dedicated to the common features of monoamine receptors, and could be considered as a hinge residue to enhance and propagate the signal from the ligand binding pocket to the cytoplasmic end of the receptor. The below Fig. 6.6 summarizes the position of the key residues discussed to participate in the conductance of the signal pathway in 5-HT2C. Implication of such pathway in monoamine receptors like 5-HT2C is supported by works (Jensen et al. 2001) which have shown agonist-induced spectral changes for preferred conformations of residues 6.33 and 6.34 in ADRB2, conserved in 5-HT2C, and suggesting an agonist-promoted movement of the cytoplasmic part of TM6 away from the receptor core and upwards toward the membrane bilayer.

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Defeat Drugs and Live Free

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