Fig. 6.

Characterization of Y284^7.55C and Y293^8.54F mutant receptors. [³⁵S]GTPγS (A) and [¹²⁵I]I-AB-MECA dissociation (B) binding assays with HEK293 cell membranes expressing Y284^7.55C or Y293^8.54F mutant receptors alone or in combination. The allosteric actions of LUF6000 were lost with either mutation, supporting the participation of π-π stacking with Y284^7.55 and Y293^8.54 and H-bonding between Y293^8.54 and the exocyclic nitrogen of LUF6000 as critical interactions for LUF6000 binding to the proposed S4 allosteric site. For the [³⁵S]GTPγS binding assays, concentration-response curves with Cl-IB-MECA were conducted in the presence of vehicle (DMSO, black) or 0.1 (yellow), 1 (blue), or 10 μM (magenta) LUF6000. Results were normalized to the E_max value of Cl-IB-MECA obtained in the presence of vehicle. The dotted line in each graph demarcates the E_max of Cl-IB-MECA in the presence of 10 μM LUF6000 with membranes from wild-type A₃ARs (240 ± 11% of vehicle). EC₅₀, E_max, and k values are reported in Supplemental Table 2. Data are presented as the mean ± S.D.