Is GPCR oligomerization required for receptor activation and G protein coupling

While the crystal structure of rhodopsin suggested that this receptor exists and operates as a monomeric structure, it is apparent that many, if not all other GPCRs might form either homodimeric or even heterodimeric complexes (see Bouvier 2001 and Chapter 1). The strongest evidence for unequivocal roles of dimerization has been obtained among the Family 3 receptors (see Part 4). The crystal structure of the extracellular domain of the metabotropic glutamate receptor unraveled not only that the receptor exists as a dimeric protein but also

Basal

Pindolol stimulated

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Pindolol stimulated

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Receptor density (pmol/mg protein)

E3.49

E6.30

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D3.49

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E6.30

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D3.49

E6.30

Fig. 3.4 Activation of the p2 adrenergic receptor involves disruption of an ionic lock between TM3 and TM6. The upper left panels show how mutation of Glu2686 30 to Gln and Ala constitutively enhances basal receptor activity. The upper right panels show how the combined

Fig. 3.4 Activation of the p2 adrenergic receptor involves disruption of an ionic lock between TM3 and TM6. The upper left panels show how mutation of Glu2686 30 to Gln and Ala constitutively enhances basal receptor activity. The upper right panels show how the combined that dimerization of the receptor most likely plays a key role in the receptor activation mechanism (Kunishima et al. 2000). Moreover, studies of the metabotropic GABAb receptors, which also belong to family C, showed yet another function of dimerization. Thus, formation of a heterodimeric complex between the GABAb1 (GBR1) and GABAb2 (GBR2) was found to be required for surface expression of a functional receptor (see Bouvier 2001). Specifically, GBR1 requires GBR2 for proper targeting to the surface, whereas GBR2 by itself is surface expressed but does not bind GABA on its own (see Bouvier 2001). Interestingly, this heterodimerization may also be critical for receptor activation and G protein coupling since recent data have provided support for a surprising model where GABA binds to GBR1 but transmission of the signal to the G protein occurs solely via GBR2 (Calver et al. 2001; Robbins et al. 2001; Duthey et al. 2001; Margeta-Mitrovic et al. 2001). How this signal transmission takes place from one receptor to the other at the structural level is unknown as well as it remains to be clarified whether a similar scenario might be relevant for other GPCRs.

The formation of homodimers among Family 1 receptors in living cells has been supported in particular by the measurements ofbioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) using fusion constructs between a GPCR and luciferase and/or yellow green fluorescent proteins, respectively (Bouvier 2001). In some cases, it has even been shown that agonists cause alterations in the degree of dimerization as reflected in changes in BRET or FRET. However, while the agonist-induced increase in energy transfer in the p2 adrenergic receptor and in the thyrotropin-releasing hormone

Fig. 3.4(Continued) mutation of Asp130349 and Glu268630 dramatically enhances the efficacy of the weak partial agonist, pindolol. It should be noted that mutation of Glu268630also enhances the efficacy of pindolol as well as mutation of Asp130349 alone both enhances basal and pindolol-stimulated activity (data not shown, see Rasmussen et al. 1999; Ballesteros et al. 2001a). However, the combined mutations cause the most dramatic effect (Ballesteros et al. 2001a). The experiments were done in transiently transfected COS-7 cells (Ballesteros et al. 2001a). The values ([3H]cAMP accumulation) are plotted as a function of receptor density, n = 4-10 individual transfections of wild-type, E268Q, E268A, D130N/E268Q, and D130N/E268A. The lower panel shows molecular 3D-representations of the interaction of TM3-TM6 at their cytoplasmic ends and the effects of 6.30 mutations. The Ca traces are taken from the high-resolution structure of bovine rhodopsin (Palczewski et al. 2000). Except in panel a, the top of each panel shows the extracellular end and the bottom the intracellular end of the TMs. In panel a, an extracellular view of the high-resolution structure of rhodopsin showing the interaction between residues at the cytoplasmic ends of TM3 and TM6 (Palczewski et al. 2000). In panel b, the Ca ribbons of TM3 and TM6 of rhodopsin are shown in purple. The simulated structures (light blue ribbons) are superimposed from position 1 to 17 onto the corresponding parts of TM6 (6.30 to 6.46) (Ballesteros etal. 2001a). The blue arrow indicates the conformational space that a Pro-kink can assume relative to TM3. Panel c shows a closer view of the interactions of Glu6.30-Arg3.50-Asp3.49 in a model of the p2 adrenergic receptor based on the rhodopsin structure. In c, the wild-type interactions are shown, with the closest contacts between Glu6.30-Arg3.50 and Arg3.50-Asp.49, which are within the distance range of an ionic interaction, shown in dashed lines. Panels d and e show the same view as in Panel c, but with alterations in putative interactions after mutation of Glu6.30 to Ala (d) or of Glu6.30 to Gln and of Asp3.49 to Asn (e). (Figure modified from Ballesteros etal. 2001, J Biol Chem 276, 29171-7). (See Plate 4.)

receptor (Angers et al. 2000; McVey et al. 2001), a decrease has recently been observed for the cholecystokinin receptor (Cheng and Miller 2001). Thus, agonist may either stabilize the dimeric complex or in some cases dissociate them. All in all, the capability of GPCRs to form dimers is now evident and in many cases a critical function has been substantiated. However, the functional implications of dimerization are surprisingly diverse and therefore there is still no general evidence among Family A receptors indicating that the formation of an oligomeric complex at the structural level is an ultimate prerequisite for receptor activation and G protein coupling. Obviously, further studies are required to clarify these aspects.

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