The Seventh Transmembrane Domains of the and Opioid Receptors
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- 作者：Wei Xu ; Mercedes Campillo ; Leonardo Pardo ; J. Kim de Riel ; and Lee-Yuan Liu-Chen
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 13, 2005
- 年：2005
- 卷：44
- 期：49
- 页码：16014 - 16025
- 全文大小：552K
- 年卷期：v.44,no.49(December 13, 2005)
- ISSN：1520-4995
文摘
We applied the substituted cysteine accessibility method (SCAM) to map the residues of thetransmembrane helices (TMs) 7 of 
and 
opioid receptors (OR and OR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in theTMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Mostmutants displayed similar binding affinity for [3H]diprenorphine, an antagonist, as the wild types.Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [3H]diprenorphine binding toeight OR and eight OR mutants. All mutants except OR L7.52(317)C were protected by naloxonefrom the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of OR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of OR are on the water-accessible surfaceof the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptorsled to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to faceTM1 are sensitive to MTSEA in OR but are not in OR. Thus, TM1 may be closer to TM7 in OR thanin OR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in OR and at 7.34(311) in OR,suggesting that TM7 in OR may have an additional helical turn (from 7.30 to 7.33). (iii) There is aconserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxPmotif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptorsin inactive states. To the best of our knowledge, this represents the first such comparison of the structuresof two highly homologous GPCRs.
and opioid receptors (OR and OR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in theTMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Mostmutants displayed similar binding affinity for [3H]diprenorphine, an antagonist, as the wild types.Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [3H]diprenorphine binding toeight OR and eight OR mutants. All mutants except OR L7.52(317)C were protected by naloxonefrom the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of OR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of OR are on the water-accessible surfaceof the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptorsled to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to faceTM1 are sensitive to MTSEA in OR but are not in OR. Thus, TM1 may be closer to TM7 in OR thanin OR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in OR and at 7.34(311) in OR,suggesting that TM7 in OR may have an additional helical turn (from 7.30 to 7.33). (iii) There is aconserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxPmotif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptorsin inactive states. To the best of our knowledge, this represents the first such comparison of the structuresof two highly homologous GPCRs.