Crosstalk in G protein-coupled receptors: Changes at the transmembrane homodimer interface determine activation

Abstract

Functional crosstalk between G protein-coupled receptors in a homo-or heterodimeric assembly likely involves conformational changes at the dimer interface, but the nature of this interface is not yet established, and the dynamic changes have not yet been identified. We have mapped the homodimer interface in the dopamine D2 receptor over the entire length of the fourth transmembrane segment (TM4) by crosslinking of substituted cysteines. Their susceptibilities to crosslinking are differentially altered by the presence of agonists and inverse agonists. The TM4 dimer interface in the inverse agonist-bound conformation is consistent with the dimer of the inactive form of rhodopsin modeled with constraints from atomic force microscopy. Crosslinking of a different set of cysteines in TM4 was slowed by inverse agonists and accelerated in the presence of agonists; crosslinking of the latter set locks the receptor in an active state. Thus, a conformational change at the TM4 dimer interface is part of the receptor activation mechanism.crosslinking ͉ cysteine ͉ dopamine ͉ oligomer ͉ rhodopsin G protein-coupled receptors (GPCRs) constitute a large superfamily of receptors that couple binding of a diverse group of ligands to activation of heterotrimeric G proteins (1). Although many GPCRs have been inferred to be dimers or oligomers in the plasma membrane (2-6), the role of GPCR oligomerization remains enigmatic. Functional interactions in heterodimeric receptor complexes have been inferred based on novel pharmacological properties and synergistic or antagonistic effects on signaling attributed to activation of each protomer (7-12). These findings are provocative but do not provide structural insights into the mechanism of this crosstalk.Another form of crosstalk that requires communication between protomers is transactivation. In the family C heterodimeric GABA B receptor, the N terminus of GB1 (but not GB2) binds GABA (13), whereas cytoplasmic loops of GB2 couple to G protein (14-16). Transactivation has also been reported for a family C metabotropic glutamate receptor and for the family A leutinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH) receptors (17)(18)(19)(20).An understanding of the structural basis of crosstalk between receptors in a dimer requires identification of the dimerization interface and its changes, but information about these interfaces is still rather limited. Based on the spatial arrangement of rhodopsin arrays visualized by atomic force microscopy (AFM) of mouse retinal disk membranes in the inactive state, Liang et al. (21) built a molecular model that features a symmetric homodimer interface involving both transmembrane segment (TM) 4 and TM5 (Fig. 1A). In contrast, an 8.4-Å 3D structure was derived from electron cryomicroscopy (ECM) of tilted 2D crystals of squid rhodopsin reconstituted from detergent into lipid (22). This structure, which was proposed to be related to the arrangement of squid rhodopsin in the native membrane, has a symmetric interfa...

Influence of oligomerization on the dynamics of G‐protein coupled receptors as assessed by normal mode analysis

“…11 Combined essential dynamics of these simulations showed that the most pronounced changes within the transmembrane region of the rhodopsin dimer occurred at the dimerization interface. Albeit consonant with the rearrangement of the dimer interface observed for the cognate dopamine D2 receptor, 10 the results of these simulations do not allow discriminating among the different dynamic mechanisms that could account for such a conformational rearrangement. In fact, calculations were limited by the system size and submicrosecond time scales accessible with current computer hardware and algorithms.…”

“…Additional cross-linking data of cysteine mutants at the D2DR homo-dimer interface in the presence of agonists and inverse agonists 10 suggested the occurrence of a conformational rearrangement of the dimer interface upon receptor activation. Specifically, agonists accelerated crosslinking of a set of residues on a different TM4 face than the one preferred by the inverse agonist, while diminished crosslinking was recorded for residues on the interface preferred by the inverse agonist.…”

“…At least three different dynamic mechanisms can account for this conformational rearrangement. 10 As viewed from the intracellular side of the membrane, these rearrangements are: (1) a simple rigid body clockwise rotation of contacting TM4s along their own helical axes [ Fig. 1(B)], (2) a displacement of the protomers [i.e.…”

Light on the structure of thromboxane A2 receptor heterodimers

“…Additional contacts involving the cytosolic ends of H3, H4 and IL2 are shown by the CXCR4 complex with the cyclic peptide [29]. For the D 2 R, cysteine crosslinking experiments interpreted in the context of the structural models highlighted the potential rearrangements of the dimer architecture depending on the receptor functional state, the H4-H5 and H4-H4 contact dimers being, respectively, associated with inactive and active states [25,26]. In a more recent study, the same authors provided evidence that D 2 R forms higher-order oligomers in living cells and that H1 and H8 form a second symmetric interface in addition to the previously identified H4 interface [16].…”

“…System's dependent variety in the dimer architecture emerges also from results of in vitro experiments. In this respect, the highest resolution information available thus far comes from X-ray crystallography and atomic force microscopy (AFM) measurements on rhodopsin, opsin, b 2 -AR and CXCR4 chemokine receptor, as well as from cysteine crosslinking experiments on D 2 receptor (D 2 R), and disulphide trapping experiments on 5HT 1c receptor [22][23][24][25][26][27][28][29]. With regard to rhodopsin, the geometrical constraints from AFM measurements led to the proposal of a semi-empirical model of a higher order rhodopsin structure [22].…”