Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

Abstract

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an ␣-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.G protein-coupled receptors (GPCRs) 3 constitute the largest family of cell-surface signal transduction proteins in the human genome. They function as plasma membrane-embedded receptors for ligands such as biogenic amines, peptides, and protein hormones. The receptor transduces the hormone binding signals across the membrane to heterotrimeric G proteins. The oligomeric state of the receptor and the stoichiometry of the receptor-G protein complex is a topic of considerable debate (1-4). Many GPCRs from each of three major classes (A, B, and C) have been shown to homo-oligomerize, and hetero-oligomerization of structurally related members of the same class has also been demonstrated (2,3,5). In many cases the specific oligomeric state of the receptor is unclear. It often is assumed to be the dimer, although evidence for higher order arrays of receptors also exists (6 -8). Ligand effects on oligomerization can vary from no effect (9) to ligand-induced oligomerization (10) or ligand-induced disruption of oligomerization (11). The receptor domains responsible for oligomerization can vary from individual helices of the 7-transmembrane (7-TM) helical domain (12) to extracellular (13) or intracellular domains (14). The most convincing argument for a functional role for oligomerization comes from studies of the class C ␥-aminobutyric acid, type B receptor (14 -16) and the sweet and umami taste receptors (17), which function as obligate heterodimers. In addition, the class A leukotriene B4 and dopamine D2 receptors have been shown to couple to G protein as dimers (18,19). These studies suggested that the pentameric complex of a GPCR dimer and a single G protein heterotrimer is the minimal functional signaling unit. However, several rigorous biochemical studies have shown that the ␤2-adrenergic receptor and rhodopsin, both class A receptors, are capable of functionally coupling to G proteins as monomers (20 -23) even though oligomerization of these receptors is well documented (6,8,24). Perhaps the only ...

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

“…For family B receptors, there is consistent evidence for homooligomerization, together with the potential for functionally important heterooligomerization (18)(19)(20)(21)(22)(23)(25)(26)(27). Within this theme, there is an emerging paradigm of behavior in which homodimerization of receptors occurs via a TM4/TM4 interface and that this interaction is required for optimal function of the receptor, generation of high-affinity agonist binding, and signaling via formation of cAMP (17,18,31); this paradigm is true also for the GLP-1R.…”

“…For family B GPCRs, which encompass many therapeutically important peptide receptors, including those for glucagon, glucagon-like peptides 1 and 2 (GLP-1, GLP-2), parathyroid hormone, and calcitonin, there is consistent evidence for homodimerization (17)(18)(19)(20)(21)(22)(23)(24)(25). There is also emerging evidence for functionally significant heterodimerization (25)(26)(27).…”

Type 2 Diabetes Mellitus and Glucagon Like Peptide-1 Receptor Signalling

“…For family B GPCRs, homodimerisation has been shown to occur with the calcitonin receptor [149)], secretin receptor [150] and parathyroid receptor [151]. There is also interest in the development of allosteric agonists and whether they interact with a single receptor (in cis) or across dimers (in trans).…”

Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation

“…A large number of fluorescent ligands are available for GPCRs (Kuder and Kiec-Kononowicz 2008), but there will no doubt be receptors for which production of such ligands is challenging. It should be noted that in this type of experiment it is impossible to study dimers in the unliganded state, a condition which may be of particular significance when ligand binding can promote or inhibit dimer formation (Lukasiewicz et al Pioszak et al 2010). Finally, it may not be possible to study receptors expressed at low levels or cells which express multiple receptors for the same ligand.…”

“…A wholecell BRET approach with PTH1R fused to C-terminal luciferase or YFP revealed that PTH1R forms a constitutive dimer which is mediated by the ECD (Pioszak et al 2010). Addition of the agonist PTH disrupted the dimer, suggesting that monomers are the signalling unit.…”