The conserved tryptophan in position 13 of TM-VI (Trp-VI:13 or Trp-6.48) of the CWXP motif located at the bottom of the main ligand-binding pocket in TM-VI is believed to function as a rotameric microswitch in the activation process of seventransmembrane (7TM) receptors. Molecular dynamics simulations in rhodopsin demonstrated that rotation around the chi1 torsion angle of Trp-VI:13 brings its side chain close to the equally highly conserved Phe-V:13 (Phe-5.47) in TM-V. In the ghrelin receptor, engineering of high affinity metal-ion sites between these positions confirmed their close spatial proximity. Mutational analysis was performed in the ghrelin receptor with multiple substitutions and with Ala substitutions in GPR119, GPR39, and the ␤ 2 -adrenergic receptor as well as the NK1 receptor. In all of these cases, it was found that mutation of the Trp-VI:13 rotameric switch itself eliminated the constitutive signaling and strongly impaired agonist-induced signaling without affecting agonist affinity and potency. Ala substitution of Phe-V:13, the presumed interaction partner for Trp-VI:13, also in all cases impaired both the constitutive and the agonist-induced receptor signaling, but not to the same degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency. In a proposed active receptor conformation generated by molecular simulations, where the extracellular segment of TM-VI is tilted inwards in the main ligand-binding pocket, Trp-VI:13 could rotate into a position where it obtained an ideal aromatic-aromatic interaction with Phe-V: 13. It is concluded that Phe-V:13 can serve as an aromatic lock for the proposed active conformation of the Trp-VI:13 rotameric switch, being involved in the global movement of TM-V and TM-VI in 7TM receptor activation.Despite the fact that the large superfamily of 7TM 3 or G protein-coupled receptors are activated by agonists of incredibly different chemical nature, it is believed that they nevertheless all share a common molecular activation mechanism (1-3). A series of biochemical and biophysical studies indicate that receptor activation is associated with relatively large overall changes in the arrangement of the seven-helical bundle of transmembrane segments (4, 5). This notion has been gathered in a unifying "global toggle switch" activation model describing how in particular TM-VI performs a "vertical" see-saw movement around a pivot in the middle of the membrane during activation (2). Thus, the extracellular segment of TM-VI is supposed to tilt into the main ligand-binding pocket, whereas the intracellular segment tilts outward, away from the receptor center and thereby allows binding of the active form of the G protein. However, changes in the relative conformation of TM-V and -VII are also supposed to be important parts of the activation process in which the conserved proline residues in the middle of the transmembrane segments are involved, as in TM-VI (2, 6). Recently, the x-ray structure of opsin in complex with ...

Five highly conserved polar residues connected by a number of structural water molecules together with two rotamer microswitches, TrpVI:13 and TyrVII:20, constitute an extended hydrogen bond network between the intracellular segments of TM-I, -II, -VI, and -VII of 7TM receptors. Molecular dynamics simulations showed that, although the fewer water molecules in rhodopsin were relatively movable, the hydrogen bond network of the ␤2-adrenergic receptor was fully loaded with water molecules that were surprisingly immobilized between the two rotamer switches, both apparently being in their closed conformation. Manipulations of the rotamer state of TyrVII:20 and TrpVI:13 demonstrated that these residues served as gates for the water molecules at the intracellular and extracellular ends of the hydrogen bond network, respectively. TrpVI:13 at the bottom of the main ligand-binding pocket was shown to apparently function as a catching trap for water molecules. Mutational analysis of the ␤2-adrenergic receptor demonstrated that the highly conserved polar residues of the hydrogen bond network were all important for receptor signaling but served different functions, some dampening constitutive activity (AsnI:18, AspII:10, and AsnVII:13), whereas others (AsnVII:12 and Asn-VII:16) located one helical turn apart and sharing a water molecule were shown to be essential for agonist-induced signaling. It is concluded that the conserved water hydrogen bond network of 7TM receptors constitutes an extended allosteric interface between the transmembrane segments being of crucial importance for receptor signaling and that part of the function of the rotamer micro-switches, TyrVII:20 and TrpVI:13, is to gate or trap the water molecules.In recent years several structures of 7TM 2 receptors have been published, making it possible to compare conserved regions in different receptors and study their putative functional importance (1-7). The most extended of these highly conserved regions is the water hydrogen bond network located between the intracellular segments of TM-I, TM-II, TM-VI, and TM-VII reaching from TrpVI:13 of the CWXP motif at the bottom of the main ligand-binding pocket to TyrVII:20 of the NPXXY motif on the intracellular side of the receptor (Fig. 1). A number of conserved polar residues in these transmembrane segments have for many years been suspected to form a hydrogen bond network of likely functional importance. The recent x-ray structures have confirmed this notion but have also revealed that very few hydrogen bonds are in fact found directly between the side chains of these polar residues. Instead, a number of structural water molecules function as integral connecting parts of the hydrogen bond network (Fig. 1). Thus, in the B2AR structure (2, 3) six internal water molecules form hydrogen bonds with the highly conserved polar amino acid residues located between TrpVI:13 and TyrVII:20, whereas in the rhodopsin structures (8 -10) only four water molecules have been identified in this network (Fig. 1). 3Several studies have...

Background: A unique Glu(2.50) in the NK1 receptor interacts directly with Ser(3.39) and Asn(7.49).

Background: Unnatural amino acids can be genetically incorporated into 7-transmembrane receptors. Results: A photoreactive amino acid introduced into the neurokinin-1 receptor cross-links substance P to the N-terminal and extracellular loop II domains of the receptor. Conclusion:The extracellular domain of the neurokinin-1 receptor possesses multiple potential binding sites for substance P. Significance: A photocross-linking methodology reveals novel interaction sites in the neurokinin-1-receptor-substance P complex.