Review
Dimerization of G protein-coupled receptors: CB1 cannabinoid receptors as an example

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Abstract

A polyclonal antibody directed towards the last 73 amino acid residues of the rat type 1 cannabinoid (CB1) receptor strongly and exclusively labels a high molecular weight (between 160 and 200 kDa) form of the receptor in Western analysis. In contrast, a human CB1 polyclonal antibody identifies both monomeric CB1 as well as the high molecular weight form. The carboxy terminus (CT) antibody was also used in immunocytochemistry of rat hippocampal sections. Sections probed with CT antibody show intense staining of a meshwork of fibers and occasional interneurons of the stratum oriens, stratum pyramidal, and stratum radiatum of the CA1 and CA3 regions while mossy fibers and granule cells of the internal stratum appear unstained. These data provide evidence that CB1 likely exists as a dimer in vivo and that the carboxy end of the receptor may play a role in the assembly of the oligomer.

Introduction

Over the past several years, it has become increasingly apparent that G-protein coupled receptors (GPCRs) can and do interact with one another to form dimers or larger oligomeric complexes (Gomes et al., 2001, Milligan, 2001). More importantly, the function of these GPCRs is very likely dependent upon the multimerization state of the protein. One of the most abundant GPCRs in the brain is the cannabinoid receptor, CB1. Once bound to agonist, CB1 activates heterotrimeric G proteins to produce downstream effects such as adenyl cyclase inhibition, MAP kinase activation, the inhibition of voltage-gated Ca2+ channels and the potentiation of inwardly rectifying potassium channels (KIR). The CB1 receptor is localized on nerve terminals and serves to regulate neurotransmitter release and neuronal plasticity (Hajos, 2000; Hoffman and Lupica, 2000, Wilson et al., 2001, Wilson and Nicoll, 2001). Expressed in many regions of the brain, including the hippocampus, cerebral cortex, basal ganglia and cerebellum (Herkenham, 1992, Tsou et al., 1998), CB1 receptors have been found to play a major role in the modulation of analgesia, body temperature, initiation of movement and mood (Ledent et al., 1999, Howlett et al., 2002). As with almost all GPCRs, the tertiary and quarternary structure of CB1 is still a mystery. However, its ability to come together with other like receptors to form larger oligomeric structures may be an important key as to how this molecule functions in vivo.

Indications for CB1 oligomerization and how this may relate to function are best viewed in the light of other descriptions of GPCR dimerization. One of the most widely cited reports of interactions between m3 muscarinic and the α2c-adrenergic receptors using several chimeras (Maggio et al., 1993). In this work, it was shown that mutant constructs of each receptor in which the carboxy ends were switched (transmembrane regions α2c I-V/m3 VI-VII or m3 I-V/α2c VI-VII) displayed little function when each chimera was individually expressed in COS-7 cells. Yet, when these receptors were co-expressed, specific ligand binding and phosphatidylinositol hydrolysis activity were observed at levels similar to those of wild type m3 receptors. Subsequent to this, both the m3 and the β2-adrenergic receptors were found to homodimerize as well (Hebert et al., 1996, Zeng and Wess, 1999, Angers et al., 2000). In the case of the β2-adrenergic receptor, it was convincingly shown that when dimer formation was disrupted by a peptide, agonist-induced adenylyl cyclase stimulation was inhibited (Hebert et al., 1996). A similar finding was reported for homodimers of the delta opioid receptor, which would monomerize upon exposure to specific agonists and would shortly thereafter be internalized (Cvejic and Devi, 1997). Later work from the same group investigated delta and kappa opioid receptors expressed in cultured cells (Jordan and Devi, 1999). In this study, it was shown that cells cotransfected with both receptors and treated with agonist specific for one or the other receptor displayed ligand-binding profiles much different from cells expressing either receptor individually. These results suggest that an important mechanism for the modulation of GPCR function is the physical interaction of one type of receptor with another. They also suggest much more complexity in terms of our understanding of how receptors work in vivo. In the past several years, many other GPCRs have been found to dimerize. The list includes dopamine (Ng et al., 1995, Lee et al., 2000), somatostatin (Rocheville et al., 2000), and thyrotropin-releasing hormone receptors (Kroeger et al., 2001). In each example, direct biochemical evidence for receptor-receptor interaction was often preceded by years of indirect data (Gomes et al., 2001).

So far, there are no direct data supporting the notion of CB1 dimerization. However, in this report we provide biochemical and immunocytochemical evidence for CB1 dimerization using a CB1 carboxy terminus-specific antibody. This antibody appears to have a high affinity for an oligomeric form of CB1.

Section snippets

Antibody production

Rabbits were injected with a fusion protein composed of glutathione-S transferase (GST) and CB1 residues 401–473 using conventional techniques. Polyclonal antiserum was collected and antibodies specific for CB1 were purified by affinity chromatography against both GST and the immunizing fusion protein.

Membrane preparation

Adult Sprague–Dawley rats were sacrificed following isofluorane anesthesia and brains were immediately homogenized in 10:1 volume:weight of 4 °C homogenization buffer (25 mM HEPES pH 7.4, 1 mM

Western blotting

Rat CB1 carboxy terminal-specific polyclonal antibody appears to display a strong affinity for a high molecular weight form of the receptor as compared with the human amino terminus CB1 antibody analyzed via Western blot (Fig. 1). As can be seen from the figure, hCB1 identifies two protein bands of around 53 and 64 kDa as well as a high molecular weight form of between 160 and 200 kDa. In contrast, the CT antibody appears to label strongly and exclusively the higher molecular weight form. No

Discussion

This report provides data supporting the notion of CB1 homo- or heterodimerization. Previously, it was shown that when isolated from rat brain and visualized on SDS-PAGE under reducing conditions, CB1 exists as either a nonglycosylated or a glycosylated monomer of approximately 53 and 64 kDa, respectively (Song and Howlett, 1995). A slower migrating form could also be seen at between 160 and 200 kDa. Our lab has developed an antibody that appears to be specific for this higher form. The

References (35)

  • C. Song et al.

    Rat brain cannabinoid receptors are N-linked glycosylated proteins

    Life Sci.

    (1995)
  • K. Tsou et al.

    Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system

    Neuroscience

    (1998)
  • R.I. Wilson et al.

    Presynaptic specificity of endocannabinoid signaling in the hippocampus

    Neuron

    (2001)
  • F.Y. Zeng et al.

    Identification and molecular characterization of m3 muscarinic receptor dimers

    J. Biol. Chem.

    (1999)
  • S. Angers et al.

    Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

    Proc. Natl. Acad. Sci. USA

    (2000)
  • M. Glass et al.

    Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor

    J. Neurosci.

    (1997)
  • I. Gomes et al.

    G protein coupled receptor dimerization: implications in modulating receptor function

    J. Mol. Med.

    (2001)
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