Section 10
Chapter 9,800

Comparison of the amino acid residues in the sixth transmembrane domains accessible in the binding-site crevices of m, d, and k opioid receptors

Xu, W.; Li, J.; Chen, C.

Biochemistry (American Chemical Society) 40(27): 18-29


Accession: 009799461

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We have mapped the residues in the sixth transmembrane domains (TMs 6) of the m, d, and k opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the m and k receptors and the wild-type d receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat m receptor and 9 of 22 mutants of the human d receptor and 10 of 22 mutants of the human k receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C m mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the m receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the d receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the k receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the m, d, and k opioid receptors are consistent with the notion that the TMs 6 are in a-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure. Reprinted by permission of the publisher.

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