Present address: Galenea Corp., Cambridge, Massachusetts (AE), Pharmaceuticals Inc., Cambridge, Massachusetts (SG) and MedImmune, Gaithersburg, Maryland (RK).
Reversed binding of a small molecule ligand in homologous chemokine receptors – differential role of extracellular loop 2
Article first published online: 10 APR 2012
© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society
British Journal of Pharmacology
Special Issue: Themed Section: Secretin Family (Class B) G Protein-Coupled Receptors - from Molecular to Clinical Perspectives. Guest Editors: David R Poyner and Debbie L Hay
Volume 166, Issue 1, pages 258–275, May 2012
How to Cite
Jensen, P., Thiele, S., Steen, A., Elder, A., Kolbeck, R., Ghosh, S., Frimurer, T. and Rosenkilde, M. (2012), Reversed binding of a small molecule ligand in homologous chemokine receptors – differential role of extracellular loop 2. British Journal of Pharmacology, 166: 258–275. doi: 10.1111/j.1476-5381.2011.01771.x
- Issue published online: 10 APR 2012
- Article first published online: 10 APR 2012
- Accepted manuscript online: 3 NOV 2011 05:14AM EST
- Received; 3 February 2011; Revised; 19 September 2011; Accepted; 26 September 2011
Figure S1 Screening for agonist activity of LMD-559 among endogenous chemokine receptors. IP3 accumulation experiments performed in transiently transfected COS-7 cells. Dose–response curves for LMD-559 and a positive control selected amongst the possible endogenous agonists for all human chemokine receptors (except for CCR1 and CCR8, which are depicted in Figure 1). In addition, CCR11 was excluded, although no activation was observed for the non-peptide compound, as no endogenous ligand exists for this receptor (n = 3).
Figure S2 Screening for antagonism by LMD-559 amongst endogenous chemokine receptors. IP3-accumulation experiments performed in transiently transfected COS-7 cells. The test for antagonism was performed by stimulation of each receptor with 10 nM of the depicted endogenous ligand. Dose–response curves for LMD-559 and a positive control are shown for the same receptors as in Figure S1 (n = 3).
Figure S3 Comparison of the minor binding pocket. The depth and expansion of the ligand-binding pocket is compared in four available crystal structures: CXCR4, Rhodopsin, β2-adrenoceptor (ADR) and adenosine A2A receptor. The top panel shows the receptors from the extracellular side, whereas the lower panel illustrates the receptors from the side as viewed from TM-VI and -VII, with the corresponding ligands shown in green sticks (It1t, retinal, carazolol and ZM241385 respectively). The binding pockets are illustrated with transparent space filling, and the minor binding pocket is highlighted with stippled lines.
Figure S4 LMD-559 modelling using the β2-adrenoceptor (ADR) as template. The molecular modelling of LMD-559 in CCR8 (A,B) and in CCR1 (C,D) is presented as seen from above (A,C) and in a side view corresponding to the view from TM. The mirrored orientation of LMD-559 is apparent also in the modelling based on β2-ADR as template. Noticeable, the same key residues are identified as using CXCR4 as template and again corresponding to the residues identified by the mutational analyses. Importantly, the different roles of ECL-2B in the two receptors are confirmed. Thus, where it, in CCR1, seems to contribute directly to the binding site of LMD-559, it has a more indirect conformational constraining role in CCR8. This is indeed also apparent from the modelling based on β2-adrenoceptor (B,D).
Figure S5 Extracellular view of small molecule binding in different receptors. The top panel display small molecule ligand binding in three different chemokine receptors (all based on the crystal structure of CXCR4). From the left: LMD-559 binding in CCR8, the mirrored binding of LMD-559 in CCR1 and It-1t in CXCR4. The lower panel displays three crystal structures solved when interacting with the small molecule ligands: retinal in Rhodopsin, carazolol in the β2-adrenoceptor (ADR) and ZM241385 in the adenosine A2A receptor. The group of structural water molecules that fill up the minor binding pocket in the A2A receptor (Jaakola et al., 2008) is depicted in red sticks.
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