Mutational Analysis of the Conserved Asp^2.50and ERY Motif Reveals Signaling Bias of the Urotensin II Receptor

Abstract

Class

Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor

“…The sulfhydryl-specific alkylating reagent MTSEA (CH 3 [38]. Briefly, 10 ml of a 1 mM peptide solution was incubated with 20 mg of IODO-GEN, 80 ml of 100 mM borate buffer (pH 8.5), and 1mCi of Na-125 I for 30 min at room temperature, and was then purified by reversed-phase HPLC on a C-18 column.…”

“…UII binds the urotensin-II receptor (UT receptor), a member of family ''A'' of the G protein-coupled receptor (GPCR) superfamily [2]. Many structural features are associated with this family such as a short N-terminus, a highly conserved residue in each transmembrane domain (TM), a (D/E)R (3.50) Y ''Ionic lock'' motif [3] at the interface between the TM3 and the second intracellular loop (ICL), a C(W/F)xP (6.50) ''rotamer toggle switch'' motif [4] in TM6, a NP (7.50) xxY ''tyrosine toggle switch'' motif in TM7, and potential serine/threonine phosphorylation sites in the cytoplasmic tail [5,6]. The UT receptor also has two cysteine residues which participate in disulfide bonding between the first and second extracellular loops (ECL) (Fig.…”

A Conserved Protonation-Induced Switch can Trigger “Ionic-Lock” Formation in Adrenergic Receptors

“…Mutational studies have indicated that some residues are essential for receptor function, and it has also been proposed that the presence of a number of conserved water molecules in the hydrophobic interhelical core of GPCRs could play a significant role. 12,13,16,30,31 Interestingly, there is only one titrable residue in the highly hydrophobic core of adrenergic receptors (Fig. 1), as well as in rhodopsin †, the widely conserved Asp (2.50).…”

“…[5][6][7][8][9][10][11][12][13][14][15][16] Among them, disruption of the salt bridge ("ionic lock") between Arg (3.50) of the (D/E)RY motif of helix III and a partially conserved Glu(6.30) of helix VI seems to be a crucial feature of the early steps of receptor activation. 5,7,8 This conformational fingerprint is present in all the crystal structures of inactive rhodopsin, [17][18][19] and, despite being absent in the crystal structures of engineered β-adrenergic receptors, it has independently been observed in different microsecond molecular dynamics (MD) simulations of reconstructed wild-type adrenergic receptors bound to inverse agonists.…”

The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation

“…This position in TMD2 is known to be crucial for G protein activation (6,33,34). Also, this residue is suggested to be a part of an intermolecular bonding network implicating interhelical interactions between TMD2 and TMD7.…”