TY - JOUR

T1 - Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5)

AU - Steen, A

AU - Sparre-Ulrich, A H

AU - Thiele, Stefanie

AU - Guo, D

AU - Frimurer, T M

AU - Rosenkilde, M M

N1 - © 2013 The British Pharmacological Society.

PY - 2014/3

Y1 - 2014/3

N2 - BACKGROUND AND PURPOSE: A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity.EXPERIMENTAL APPROACH: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation.KEY RESULTS: [L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and β-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist.CONCLUSIONS AND IMPLICATIONS: The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the β2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.

AB - BACKGROUND AND PURPOSE: A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity.EXPERIMENTAL APPROACH: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation.KEY RESULTS: [L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and β-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist.CONCLUSIONS AND IMPLICATIONS: The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the β2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.

KW - Amino Acid Sequence

KW - Animals

KW - COS Cells

KW - Cercopithecus aethiops

KW - Enzyme-Linked Immunosorbent Assay

KW - Humans

KW - Ion Channel Gating

KW - Isoleucine

KW - Molecular Sequence Data

KW - Receptors, CCR5

KW - Sequence Homology, Amino Acid

U2 - 10.1111/bph.12553

DO - 10.1111/bph.12553

M3 - Journal article

C2 - 24328926

VL - 171

SP - 1566

EP - 1579

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

IS - 6

ER -