PMC:4504005 / 17682-19211 JSONTXT

{"project":"TEST0","denotations":[{"id":"25982113-85-93-8319023","span":{"begin":1053,"end":1057},"obj":"[\"24199627\", \"24199627\", \"24199627\"]"}],"text":"Validation of the Structures for Intact CGRP and AM1 Receptors in Cells\nTo validate the structures, we constructed several Ala substitution mutants in the CLR and RAMP2 ECDs, and they were analyzed for their effects on peptide-stimulated cAMP formation in COS-7 cells. For the CGRP receptor, Ala substitution of CLR W69, D70, K103, or Y91 significantly reduced CGRP potency, likely due to the structural roles of these residues (Figure 4, Table S3). Ala substitution of CLR W72, F92, D94, F95, H114, R119, W121, T122, or Y124, which are contacted by CGRPmut in the structure, reduced potency of the full-length CGRP \u003e20-fold with the CLR D94A mutant being strikingly defective.\nFor the AM1 receptor, Ala substitution of CLR W72, F92, F95, W121, or Y124, which are contacted by AM in the structure, resulted in \u003e40-fold decreases in AM potency (Figure 5A, Table S4). CLR D94, H114, R119, or T122 mutants were less deleterious with 4- to 9-fold decreases in AM potency. Mutation of RAMP2 E101 yielded 26-fold reduced AM potency (Table S4; Watkins et al., 2014). Surprisingly, Ala substitution of RAMP2 R97 or E105 did not affect AM signaling potency despite their contacts with AM (Figure 5B). RAMP2 R97A/E101A and E101A/E105A double mutants had defects similar to the E101A single mutant. These data emphasize that RAMP2 E101 provides the crucial contacts to AM. The Ala substitutions did not significantly alter receptor cell surface expression levels other than reduced AM1 receptor expression with CLR Y124A (Tables S3 and S4)."}

{"project":"2_test","denotations":[{"id":"25982113-24199627-118216125","span":{"begin":1053,"end":1057},"obj":"24199627"},{"id":"25982113-24199627-118216125","span":{"begin":1053,"end":1057},"obj":"24199627"},{"id":"25982113-24199627-71076120","span":{"begin":1053,"end":1057},"obj":"24199627"},{"id":"25982113-24199627-71076120","span":{"begin":1053,"end":1057},"obj":"24199627"}],"text":"Validation of the Structures for Intact CGRP and AM1 Receptors in Cells\nTo validate the structures, we constructed several Ala substitution mutants in the CLR and RAMP2 ECDs, and they were analyzed for their effects on peptide-stimulated cAMP formation in COS-7 cells. For the CGRP receptor, Ala substitution of CLR W69, D70, K103, or Y91 significantly reduced CGRP potency, likely due to the structural roles of these residues (Figure 4, Table S3). Ala substitution of CLR W72, F92, D94, F95, H114, R119, W121, T122, or Y124, which are contacted by CGRPmut in the structure, reduced potency of the full-length CGRP \u003e20-fold with the CLR D94A mutant being strikingly defective.\nFor the AM1 receptor, Ala substitution of CLR W72, F92, F95, W121, or Y124, which are contacted by AM in the structure, resulted in \u003e40-fold decreases in AM potency (Figure 5A, Table S4). CLR D94, H114, R119, or T122 mutants were less deleterious with 4- to 9-fold decreases in AM potency. Mutation of RAMP2 E101 yielded 26-fold reduced AM potency (Table S4; Watkins et al., 2014). Surprisingly, Ala substitution of RAMP2 R97 or E105 did not affect AM signaling potency despite their contacts with AM (Figure 5B). RAMP2 R97A/E101A and E101A/E105A double mutants had defects similar to the E101A single mutant. These data emphasize that RAMP2 E101 provides the crucial contacts to AM. The Ala substitutions did not significantly alter receptor cell surface expression levels other than reduced AM1 receptor expression with CLR Y124A (Tables S3 and S4)."}