Analysis of the Third Transmembrane Domain of the Human Type 1 Angiotensin II Receptor by Cysteine Scanning Mutagenesis

Abstract

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT 1 ) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile 103 -Tyr 127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT 1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT 1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT 1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT 1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

“…The basal activity of N111G-AT1R can be inhibited by AT1R-selective blockers, albeit at 3-315-fold higher concentrations compared with wild-type AT1R (16,19,20,22,23). The conformation of the TMD of the N111G-AT1R has been shown to be altered to the activated state (12,13,(32)(33)(34)(35)(36). The RCAM results in Fig.…”

Structural determinants for binding, activation, and functional selectivity of the angiotensin AT1 receptor

“…1). During this process, the receptor undergoes a series of conformational rearrangements involving rotation and movement of different mostly hydrophobic transmembrane helices, creating a water-accessible crevice that forms the binding pocket for water-soluble ligands (Martin et al 2004). Movements of TM helices are a mechanism by which the AT 1 receptor is activated, and inhibition of movements abolishes G protein activation.…”

“…Each ligand may induce rotation of helices but differs in the extent of rotation (Miura et al 2012a). This involves transmembrane movement that takes the form of a slight clockwise rotation of TMD2 (Miura & Karnik 2002), counterclockwise rotation of TMD3 (Martin et al 2004, Petrel & Clauser 2009) and TMD6 (Petrel & Clauser 2009), and lateral movement of TMD7 (Boucard et al 2003) (Fig. 1).…”

“…A slight counterclockwise rotation of TMD3 (Fig. 1) exposes residues Ala104, Ser109, Asn111, Leu112, and Val116 of TMD3 toward the ligand-binding pocket (Martin et al 2004). Mutation of Asn111 or Ser115 does not alter the binding affinity for peptide analogs, but modifies the binding of non-peptide AT 1 and AT 2 receptor antagonists.…”

“…This suggests a complex regulatory role for Asn111 side chain in the AT 1 receptor activation (Feng et al 1998). Martin et al (2004) have systematically analyzed all residues within the third TMD (Ile103-Tyr127) of the human AT 1 receptor that contribute to the formation of the ligand-binding pocket, which extends from the extracellular surface of the receptor to the transmembrane portion. Side chains of several residues such as Lys102, and from Ser105 to Val116 of TMD3, are involved in interactions with Ang II as their mutation alters receptor affinity for Ang II and its analogs (Balmforth et al 1997, Groblewski et al 1997, Feng et al 1998, Miura et al 1999.…”