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      Mechanism of Constitutive Activation of the AT 1 Receptor: Influence of the Size of the Agonist Switch Binding Residue Asn 111

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      Biochemistry

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          Abstract

          The AT 1 receptor is a G-protein-coupled receptor (GPCR); its activation from the basal state (R) requires an interaction between Asn 111 in transmembrane helix III (TM-III) of the receptor and the Tyr 4 residue of angiotensin II (Ang II). Asn 111 to Gly 111 mutation (N111G) results in constitutive activation of the AT 1 receptor (Noda et al. (1996) Biochemistry, 35, 16435–16442). We show here that replacement of the AT 1 receptors TM-III with a topologically identical 16-residue segment (Cys 101-Val 116) from the AT 2 receptor induces constitutive activity, although Asn 111 is preserved in the resulting chimera, CR18. Effects of CR18 and N111G mutations are neither additive nor synergistic. The conformation(s) induced in either mutant mimics the partially activated state (R′), and transition to the fully activated R* conformation in both no longer requires the Tyr 4 of Ang II. Both the R state of the receptor and the Tyr 4 Ang II dependence of receptor activation can be reinstated by introduction of a larger sized Phe side chain at the 111 position in CR18, suggesting that the CR18 mutation generated an effect similar to the reduction of side chain size in the N111G mutation. Consistently in the native AT 1 receptor, R′ conformation is generated by replacement with residues smaller but not larger than the Asn 111. However, size substitution of several other TM-III residues in both receptors did not affect transitions between R, R′, and R* states. Thus, the property responsible for Asn 111 function as a conformational switch is neither polarity nor hydrogen bonding potential but the side chain size. We conclude that the fundamental mechanism responsible for constitutive activation of the AT 1 receptor is to increase the entropy of the key agonist-switch binding residue, Asn 111. As a result, the normally agonist-dependent R → R′ transition occurs spontaneously. This mechanism may be applicable to many other GPCRs.

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          Author and article information

          Journal
          0370623
          1028
          Biochemistry
          Biochemistry
          Biochemistry
          0006-2960
          1520-4995
          15 September 2020
          10 November 1998
          21 September 2020
          : 37
          : 45
          : 15791-15798
          Affiliations
          Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
          Author notes
          [‡]

          These authors contributed equally to this work.

          [* ]Corresponding author. Address: Department of Molecular Cardiology/FF30, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. (216) 444-1269. FAX: (216) 444-9263. karniks@ 123456cesmtp.ccf.org .
          Article
          PMC7505089 PMC7505089 7505089 nihpa156481
          10.1021/bi980863t
          7505089
          9843384
          548bf521-32bc-4d6b-961f-a2dea03f22d4
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