Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB₂ Cannabinoid Receptor

Abstract

Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB 2 cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB 1 and CB 2 cannabinoid receptors but not in many other GPCRs) of the human CB 2 receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB 2 receptor.

By Interacting with the C-terminal Phe of Apelin, Phe255 and Trp259 in Helix VI of the Apelin Receptor Are Critical for Internalization

“…Conversion of Trp 152 to Ala or Phe led to misfolded proteins that were not able to bind pE13F, suggesting that Trp 152 is essential for apelin receptor structure integrity. This is in line with the data obtained with different G proteincoupled receptors, such as CB2 cannabinoid, M3 muscarinic, and ␦-opioid receptors, in which the mutations of the equivalent residues led to a decrease in the binding of their respective ligands (42,48,57). However, these substitutions led to less important changes than those we observed for the apelin receptor.…”

“…Clusters of TM aromatic residues have already been identified as an important moiety for G protein-coupled receptors functioning (42)(43)(44)(45)(46). In such clusters, particular interest has been taken in the tryptophan (47)(48)(49) and phenylalanine (50 -53) side chains found in TM helices, which were demonstrated as key residues for ligand binding and/or receptor activation. In biological systems, -interactions have been established as bringing an important contribution to the stability and flexibility of molecular associations (54,55).…”

Mutation Studies of Ser7.39 and Ser2.60 in the Human CB1Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB1Transmembrane Helix 7

“…In the CB 2 receptor, mutation of the homologous Ser285 (Ser7.39) residue has been reported to decrease the binding affinity of the classic agonist HU-243 (Rhee, 2002). However, the ability of HU210, CP55,940, and WIN55,212-2 to inhibit adenyl cyclase was similar to that of wild type.…”

“…In contrast, the author reports Ser292 (Ser7.46) to be involved in the binding of HU210 and CP55,940 and not WIN5512-2 via a hydrogen-bonding interaction. Furthermore, Rhee (2002) measured IC 50 value as a measure of the binding affinity, a value that intrinsically depends on the agonist concentration that is employed. The use of inhibitory constant (K i ) value (see Materials and Methods) instead should give a better estimate of ligand affinity.…”

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

“…The obtained pure CB2 fragment was characterized by circular dichroism and fluorescence spectroscopy. It has been known from the reported mutation and function studies that Ser 285 , Ser 292 and Tyr 299 in TM7 are important for CB2 ligand binding [17,18]. The residue Tyr 299 in TM7, as well as the conserved cysteins Cys 313 and Cys 320 in the C-terminal juxamembrane region were shown to be critical for coupling to adenylate cyclase [17].…”