Mutations. The conserved residues in the NxxxNPxxY motif also seem to be very sensitive to mutation considering the behavior of the point mutants and our previous random mutagenesis results that did not identify any gain-of-function mutant by mutations in this region (Beukers et al., 2004a). Typically, homology models are based on the alignment of entire helical regions between rhodopsin and receptors with unknown structure. Loop areas are normally ignored or modeled based on databases of loop conformations. These models have been used to design experiments as well as to explain experimental data. We used the structure of bovine rhodopsin as a template to build a homology model of the human adenosine A2B receptor to guide the selection of amino acids for site-directed mutagenesis. The recent disclosure of the 3-D structure of the human β2 adrenergic receptor enabled us to make a homology model based on this template too. A structural overlay was made to investigate the differences between the two models with respect to the regions of interest in this study. The following differences were identified, i) the angle between helix 7 and 8, ii) the number of residues to link helix 7 and 8, iii) the location and side chain orientation of residues in the NxxxNPxxY motif, and iv) the nature of the salt bridge between helix 1 and helix 7. These findings suggest that care should be taken to interpret experimental data based on a single model. Cherezov already pointed to the shortcoming of homology models generated from a single structural template, as the structural divergence between two receptors would be quite difficult to predict accurately using only one receptor as a template (Cherezov et al., 2007). His conclusion was drawn from a similarity comparison between the β2 adrenergic receptor crystal structure and homology models of the β2 adrenergic receptor based on the structure of bovine rhodopsin and which were substantiated with biochemical data (Bissantz et al., 2003; Furse and Lybrand, 2003; Gouldson et al., 2004). Since the latter models used bovine rhodopsin as a template, they are more similar to the rhodopsin rather than the real β2 adrenergic receptor structure. Although all class A receptors share a similar backbone consisting of 7 transmembrane domains linked by extracellular and intracellular loops, each receptor has its own characteristics. Slight differences in the structure may result in significant differences in intramolecular interactions. For example, in rhodopsin, E6.30 forms an ionic bond with R3.50 of the conserved D(E)RY motif (Palczewski et al., 2000). This interaction is postulated to be important for maintaining rhodopsin in the inactive state, but the charged groups of the two residues R3.50 and E6.30 are too far apart to form an ionic bond in the structure of the β2 adrenergic receptor-T4 lysozyme fusion protein (Cherezov et al., 2007). In the present study, mutations in the human adenosine A2B receptor NxxxNPxxY motif were made equivalent to LH receptor and 5-HT2C receptor mutations, however with strikingly different results. Apparently, the NxxxNPxxY network in the A2B receptor functions in a different manner compared to the network in the LH and the 5-HT2C receptors (Table 4). The potential salt bridge is also atypical with respect to other adenosine receptors since the E1.39H mutant is irresponsive to agonists whereas the H7.42E mutant is not expressed at all. These results suggest that the human adenosine A2B receptor NxxxNPxxY network and the potential salt bridge are optimized for receptor function of this receptor subtype and every receptor may be slightly different in this respect.
Appears in 1 contract
Sources: Doctoral Thesis
Mutations. The conserved residues in the NxxxNPxxY motif also seem to be very sensitive to mutation considering the behavior of the point mutants and our previous random mutagenesis results that did not identify any gain-of-function mutant by mutations in this region (Beukers et al., 2004a). Typically, homology models are based on the alignment of entire helical regions between rhodopsin and receptors with unknown structure. Loop areas are normally ignored or modeled based on databases of loop conformations. These models have been used to design experiments as well as to explain experimental data. We used the structure of bovine rhodopsin as a template to build a homology model of the human adenosine A2B receptor to guide the selection of amino acids for site-directed mutagenesis. The recent disclosure of the 3-D structure of the human β2 þ2 adrenergic receptor enabled us to make a homology model based on this template too. A structural overlay was made to investigate the differences between the two models with respect to the regions of interest in this study. The following differences were identified, i) the angle between helix 7 and 8, ii) the number of residues to link helix 7 and 8, iii) the location and side chain orientation of residues in the NxxxNPxxY motif, and iv) the nature of the salt bridge between helix 1 and helix 7. These findings suggest that care should be taken to interpret experimental data based on a single model. Cherezov already pointed to the shortcoming of homology models generated from a single structural template, as the structural divergence between two receptors would be quite difficult to predict accurately using only one receptor as a template (Cherezov et al., 2007). His conclusion was drawn from a similarity comparison between the β2 þ2 adrenergic receptor crystal structure and homology models of the β2 þ2 adrenergic receptor based on the structure of bovine rhodopsin and which were substantiated with biochemical data (Bissantz et al., 2003; Furse and Lybrand, 2003; Gouldson et al., 2004). Since the latter models used bovine rhodopsin as a template, they are more similar to the rhodopsin rather than the real β2 þ2 adrenergic receptor structure. Although all class A receptors share a similar backbone consisting of 7 transmembrane domains linked by extracellular and intracellular loops, each receptor has its own characteristics. Slight differences in the structure may result in significant differences in intramolecular interactions. For example, in rhodopsin, E6.30 forms an ionic bond with R3.50 of the conserved D(E)RY motif (Palczewski et al., 2000). This interaction is postulated to be important for maintaining rhodopsin in the inactive state, but the charged groups of the two residues R3.50 and E6.30 are too far apart to form an ionic bond in the structure of the β2 þ2 adrenergic receptor-T4 lysozyme fusion protein (Cherezov et al., 2007). In the present study, mutations in the human adenosine A2B receptor NxxxNPxxY motif were made equivalent to LH receptor and 5-HT2C receptor mutations, however with strikingly different results. Apparently, the NxxxNPxxY network in the A2B receptor functions in a different manner compared to the network in the LH and the 5-HT2C receptors (Table 4). The potential salt bridge is also atypical with respect to other adenosine receptors since the E1.39H mutant is irresponsive to agonists whereas the H7.42E mutant is not expressed at all. These results suggest that the human adenosine A2B receptor NxxxNPxxY network and the potential salt bridge are optimized for receptor function of this receptor subtype and every receptor may be slightly different in this respect.
Appears in 1 contract
Sources: Doctoral Thesis