Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

Abstract

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3-17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, Urotensin II–Related Peptide, and Their Receptor: From Structure to Function

“…The presence of a binding site in rat UII ECL3 is confirmed by photolabeling data (Holleran et al, 2007). However, it is not yet established whether this is a transient surface interaction that precedes a deeper set of interactions into the TMD bundle leading to receptor activation.…”

Mutational Analysis of the Conserved Asp2.50and ERY Motif Reveals Signaling Bias of the Urotensin II Receptor

“…The most conserved residues of each TMD are circled in bold. Residues of the UT receptor that are important for UII binding are in dark gray (Boucard et al, 2003;Holleran et al, 2007), and those that are important for internalization are in black with white lettering (Proulx et al, 2005). Putative Asn-glycosylation sites (Asn UT-Dependent Activation of ERK1/2 Involves Both PKC and Transactivation of the EGF Receptor.…”

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

“…Out of the top five models predicted by I-TASSER, we selected the model that was the most appropriate to allow the insertion of UII in the rUT receptor binding pocket (model 1, C-score = 0.24). Previous modeling done by us was used as the basis for the initial UII peptide orientation within the binding pocket [34,42]. The GROMACS software suite [43] was used with the OPLS/AA forcefield [44][45][46] and GBSA implicit solvent model [47] to perform potential energy minimization and a molecular dynamic simulation of the UII/rUT receptor complex.…”

“…The GROMACS software suite [43] was used with the OPLS/AA forcefield [44][45][46] and GBSA implicit solvent model [47] to perform potential energy minimization and a molecular dynamic simulation of the UII/rUT receptor complex. Distance restraints based on previous photolabeling results [42,48] and docking experiments [49] were used to guide and orient UII docking inside the binding pocket. The distance restraints were implemented in the topology file using r0 = 0 Å , r1 = 7 Å and r2 = 12 Å between the Cb atoms of the corresponding residues, with a force constant of 3000 kJ/mol/ nm 2 .…”