An intriguing suggestion from the structure of β2
AR was the ability of cholesterol and palmitate to act in concert to mediate GPCR dimer formation [3
]. It is possible that this effect was largely observed due to crystal packing conditions but such an arrangement could influence GPCR activity in vivo
The recent study by Zheng et al
] systematically investigates the effects of cholesterol and palmitoylation on OPRM1 dimerization and G protein coupling using a variety of colocalization techniques. Cholesterol can be depleted by statins and Zheng et al
. used the synthetic statin simvastatin to probe the effects of cholesterol on OPRM1. Simvastatin reduced the amount of cholesterol associated with OPRM1 but also decreased receptor dimerization and receptor-G protein interactions. The authors also demonstrate that mutagenesis of the palmitoylated cysteine residue in OPRM1 has no effect on ligand binding but does decrease signaling efficiency, probably by impairing GPCR-G protein association. The same mutant had significantly reduced dimerization, and it was proposed that this was responsible for the reduced G protein coupling. Although it has been suggested that a G protein heterotrimer is too large to signal via a monomeric GPCR, monomeric receptors have been shown to signal efficiently [8
], although this ability may be receptor-specific. Finally, Zheng et al
. demonstrate that the palmitate-free mutant associated more weakly with cholesterol [4
], suggesting a functional interaction between the palmitate group and cholesterol.
It is interesting to note that, in contrast to the β2
AR crystal structure [3
], the palmitate group on OPRM1 is not located on the carboxy-terminal tail but instead on the intracellular side of transmembrane domain (TM) 3 [4
]. In β2
AR, the dimer interface involves TM1 and helix 8 (in the carboxy-terminal tail) whereas the OPRM1 dimer interface is predicted to be between TM4 of each protomer, with the palmitate bound to the carboxy-terminal side of TM3. This difference in receptor interface may be driven by the location of palmitoylation and it is possible that regulation of palmitoylation states could dynamically influence the dimerization interfaces of GPCRs. A model of the OPRM1 dimer in which cholesterol and palmitate pack together to facilitate receptor dimerization reveals that cholesterol interactions contribute approximately 25% of the total interaction energy at the homodimer interface [4
]. Dimerization of OPRM1 appears to be regulated by palmitate, which recruits cholesterol to stabilize the dimer. Most GPCR dimers are transient, and anything that affects the weak association between protomers may have dramatic effects on the population of dimers at equilibrium.
The complex interplay between receptor, cholesterol and palmitate illustrated by Zheng et al
] lends support to the model suggested by the crystal structure of β2
] by demonstrating a role for sterols and lipids in GPCR dimerization in vivo
. It remains to be seen if other GPCRs require such modulation to support dimerization. For example, neurotensin receptor 1 can dimerize in artificial lipid membranes even in the absence of palmitoylation [9
] and also in low concentrations of detergent (no lipid bilayer) [10
]. An interesting discussion remains - what truly constitutes a GPCR dimer? How close do the receptors have to be to functionally interact? If a dimer is required solely to provide sufficient surface area for G protein coupling, then the proximity provided by such a lipid bridge seems sufficient, but if allosteric regulation by protomers within the dimer is to occur, more direct protein-protein interactions may be required.
These studies remind us that it is vital to consider membrane proteins in the context of their environment. They do not exist in isolation and are heavily influenced by lipids, sterols and other proteins. Cholesterol and palmitoylation can affect all aspects of GPCR activity and their precise roles are likely to be receptor-dependent, although only with more data can we hope to determine any consensus. Membrane environments are challenging to control and probe, but such studies are vital if we are to gain a complete understanding of GPCR function.