Cannabinoid receptor homo- and heterodimerization

Abstract

CB1 cannabinoid receptors mediate the psychoactive effects of Δ⁹THC and actions of the endogenous cannabinoids [Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A., Felder, C.C., Herkenham, M., Mackie, K., Martin, B.R., Mechoulam, R., Pertwee, R.G., 2002. International Union of Pharmacology: XXVII. Classification of cannabinoid receptors. Pharmacological Reviews 54 (2) 161–202.]. CB1 receptors belong to the G protein-coupled receptor (GPCR) superfamily. In recent years, it has become apparent that many GPCRs exist as multimers—either of like or unlike receptors [Kroeger, K.M., Pfleger, K.D., Eidne, K.A., 2003. G-protein coupled receptor oligomerization in neuroendocrine pathways. Frontiers of Neuroendocrinology 24 (4) 254–278; Milligan, G., 2004. G protein-coupled receptor dimerization: function and ligand pharmacology. Molecular Pharmacology 66 (1) 1–7.]. Importantly, GPCR multimerization plays a key role in enriching the signaling repertoire of these receptors. In this review, the evidence for CB1 multimerization will be presented, the implications for cannabinoid signaling discussed, and possible future directions for this research considered.

Introduction

Classically, G protein-coupled receptors (GPCRs) were thought to be expressed as monomers and that each individual GPCR signaled through its own pool of G proteins. Recent work from a number of investigators clearly shows this is not the case. (Review of early studies investigating binding cooperativity and radiation inactivation also support the concept of functional GPCRs as multimers.) These studies have shown that GPCRs can exist and function as dimers or higher order multimers. (Throughout this review, dimerization and multimerization are used interchangeably in deference to our incomplete knowledge of the tertiary structure of the receptor complexes.) Most dimerization studies have used immunoprecipitation (Jordan and Devi, 1999), complementation (Scarselli et al., 2001), or resonance energy transfer (Canals et al., 2004) approaches to demonstrate association.

Typically, for the immunoprecipitation studies, two receptors with distinct epitope tags are heterologously expressed in cell lines. The cells are then lysed and one receptor is immunoprecipitated with antibodies directed against its first epitope tag. The immunoprecipitated sample is then subjected to Western blotting with an antibody directed against the second tag. If the appropriate controls are conducted, and the second antibody recognizes the tagged receptor in the immunoprecipitate, then this is taken for evidence of association of the two receptors.

In complementation studies, two receptors are expressed (either with point mutations or truncations) that are by themselves inactive. If co-expression of the two receptors together reconstitutes a functional receptor, this is taken as evidence that the two proteins are interacting. An interesting feature of these complementation studies is that they have identified dominant negative or dominant positive interactions between pairs of GPCRs. The best known example of this is the interaction between CCR5 and its naturally occurring truncation CCR5D32 (Kazmierski et al., 2002). Expression of the truncated CCR5 dramatically slows wild-type CCR5 internalization (necessary for infection by M-tropic HIV).

The resonance energy transfer approaches, typically FRET (fluorescent resonance energy transfer) or BRET (bioluminescence resonance energy transfer), rely on the observation that non-radiative transfer of light between donor and acceptor molecules is proportional to the reciprocal of the separating distance raised to the sixth power (Jares-Erijman and Jovin, 2003). Thus, receptors whose donor and acceptors are close to one another (< 50–100 Å) will undergo resonance transfer, while more distant receptors will not. In FRET, the donor and acceptor molecules are coupled to the receptors (for example, by the incorporation of YFP and CFP in the primary structure of the receptors). For BRET, the enzyme luciferase is incorporated into one receptor (which will catalyze the conversion of coelenterazine to coelenteramide with the generation of light with a peak wavelength of ∼ 470 nm) (Pfleger and Eidne, 2003). YFP in the second receptor excited by the light generated during the degradation of coelenterazine will then fluoresce with a peak of ∼ 530 nm. An advantage of BRET is that no excitation of the donor is necessary, thus avoiding the problems associated with exposing living cells to relatively high-intensity, low-wavelength light such as autofluorescence, photobleaching, and phototoxicity. A drawback of BRET is that with current instrumentation and techniques, spatial resolution is poor. In contrast, with optimal conditions, FRET can be detected in subcellular domains. For the resonance energy transfer techniques, it is important to consider that not only the distance, but also the relative orientations of the donor and acceptor are important, particularly for FRET experiments. Thus, decreases in FRET in GPRC dimerization studies could be due to either an increase in the distance separating the receptors or due to the assumption of an unfavorable conformation between donor and the acceptor, in the absence of a change in the absolute separation. Thus, the most robust results will come from studies that use independent methods to determine receptor association.

By now, a great number of studies have been conducted using these techniques investigating many GPCRs. While there is some discrepancy between various studies, several common themes have emerged:

• GPCR homodimerization is very common; likely all GPCRs will be able to form homodimers.

• GPCR heterodimerization also occurs, both between receptors that are members of the same class as well as between members of different classes.

• GPCR heterodimerization can lead to binding sites that bind ligands recognized by neither receptor when expressed individually.

• GPCR heterodimerization can give rise to receptor complexes that signal and/or traffic differently from the component GPCRs.

• There is no single “dimerization domain.” Dimerization occurs via extracellular domains, transmembrane helices, and intracellular domains, depending on the receptor.

• GPCRs first associate during receptor synthesis. Furthermore, the degree and regulation of dimerization by agonists at the cell surface is quite variable among receptors.

• GPCRs can also associate with ionotropic receptors, leading to alterations in the function of both receptors.