Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May 7:3:62.
doi: 10.3389/fendo.2012.00062. eCollection 2012.

Receptor oligomerization in family B1 of G-protein-coupled receptors: focus on BRET investigations and the link between GPCR oligomerization and binding cooperativity

Sarah Norklit Roed  1 , Anne Orgaard, Rasmus Jorgensen, Pierre De Meyts
Affiliations

Receptor oligomerization in family B1 of G-protein-coupled receptors: focus on BRET investigations and the link between GPCR oligomerization and binding cooperativity

Sarah Norklit Roed et al. Front Endocrinol (Lausanne). .

Abstract

The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug _targets. During the last two decades, GPCR oligomerization has been extensively studied using methods like bioluminescence resonance energy transfer (BRET) and today, receptor-receptor interactions within the GPCR superfamily is a well-established phenomenon. Evidence of the impact of GPCR oligomerization on, e.g., ligand binding, receptor expression, and signal transduction indicates the physiological and pharmacological importance of these receptor interactions. In contrast to the larger and more thoroughly studied GPCR subfamilies A and C, the B1 subfamily is small and comprises only 15 members, including, e.g., the secretin receptor, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality in family B1 GPCRs. One example of the functional effects of GPCR oligomerization is the facilitation of allosterism including cooperativity in ligand binding to GPCRs. Here, we review the currently available data on family B1 GPCR homo- and heteromerization, mainly based on BRET investigations. Furthermore, we cover the functional influence of oligomerization on ligand binding as well as the link between oligomerization and binding cooperativity.

Keywords: BRET; GPCRs; binding cooperativity; family B1; oligomerization.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The two-step model of peptide ligand binding to family B1 GPCRs. The binding of one ligand to one family B1 GPCR occurs in two steps. In step (A) the C-terminal portion of the ligand (blue) binds to the N-terminal domain of the receptor with high affinity and specificity. In step (B), the N-terminal of the ligand (green) binds to the juxtamembrane domain of the receptor. This activates the receptor and mediates intracellular interaction with G-proteins [step (C)]. Modified from Hoare (2005).
Figure 2
Figure 2
Bioluminescence resonance energy transfer saturation studies on homo- and heteromerization of GLP-1R and GCGR. These BRET studies were performed as BRET2 saturation assays, which uses a spectrally improved form of the luciferase enzyme substrate (coelenterazine), DeepBlueC, as well as a modified form of the energy acceptor, GFP2 (Pfleger and Eidne, 2006). In addition, a mutationally improved energy donor, Rluc8, with increased stability and quantum yield was used (De et al., 2007). HEK293 cells were transiently co-transfected with a constant amount of either GCGR-Rluc8 or GLP-1R-Rluc8 and an increasing amount of either GCGR-GFP2 or GLP-1R-GFP2 and incubated for 48 h at 37°C and 5% CO2. Subsequently, the cells were harvested, diluted to a concentration of 1 × 106 cells/ml, and stimulated with either 105 nM GLP-1, 83.5 nM glucagon, or buffer (negative control) for 5 min at room temperature. The BRET2 signal was measured upon addition of the Rluc8 substrate, DeepBlueC in a Mithras plate reader. All data are plotted as the BRET2 level as a function of the GFP2/Rluc8 ratio and fitted to a one-site specific binding model in GraphPad Prism. A saturating curve indicates a specific interaction between the Rluc- and GFP2-tagged receptor. The data represent mean ± SD of three independent experiments carried out in quadruplicates. (A) GLP-1R-Rluc8/GLP-1R-GFP2 homomerization in the presence (blue) or the absence (gray) of 105 nM GLP-1 (Roed, 2011). (B) GCGR-Rluc8/GCGR-GFP2 homomerization in the presence (green) or the absence (gray) of 83.5 nM glucagon (Orgaard, 2011). (C) GCGR-Rluc8/GLP-1R-GFP2 heteromerization in the presence of either 105 nM GLP-1 (blue), 83.5 nM glucagon (green), or buffer (gray) (Orgaard, 2011).
Figure 3
Figure 3
Dissociation experiment investigating negative cooperativity in the binding of GLP-1 and glucagon to the GLP-1R and the GCGR, respectively. These dissociation assays were carried out as described by De Meyts et al. (1973) for the insulin receptor. In this procedure, a small fraction of the surface expressed receptors are pre-occupied by 125I-labeled ligand in an initial association step followed by dissociation in an “infinite dilution” of either buffer of buffer containing an excess of unlabeled ligand. Accelerated dissociation of 125I-labeled ligand in the presence of unlabeled ligand indicates the presence of negative binding cooperativity. The data are plotted as the logarithm of bound/bound0 as a function of time in minutes and fitted to a two-site exponential decay model in GraphPad Prism. All data represent mean ± SD of three independent experiments carried out in duplicates. (A) Dissociation of GLP-1 from the GLP-1R. A concentration of 5 × 106 cells/ml BHK cells stably transfected with the GLP-1R were incubated with 150,000 cpm 125I-labeled GLP-1 for 3 h at 15°C. Subsequently, the unbound 125I-GLP-1 was aspired and the cells were diluted 1:40 in either HEPES binding buffer (gray) or HEPES binding buffer with 167 nM unlabeled GLP-1 (blue) and incubated at 25°C allowing ligand dissociation for up to 1440 min (24 h) (Roed, 2011). (B) Dissociation of glucagon from the GCGR. A concentration of 5 × 106 cells/ml BHK cells stably transfected with the GCGR were incubated with 150,000 cpm 125I-labeled glucagon for 1 h at 15°C. Subsequently, the unbound 125I-glucagon was removed and the cells were diluted 1:40 in either HEPES binding buffer (gray) or HEPES binding buffer with 167 nM unlabeled glucagon (green) and incubated at 25°C allowing ligand dissociation for up to 180 min (3 h) (Orgaard, 2011).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. AbdAlla S., Lother H., el M. A., Quitterer U. (2001). Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nat. Med. 7, 1003–100910.1038/nm0901-1003 - DOI - PubMed
    1. Albizu L., Cottet M., Kralikova M., Stoev S., Seyer R., Brabet I., Roux T., Bazin H., Bourrier E., Lamarque L., Breton C., Rives M. L., Newman A., Javitch J., Trinquet E., Manning M., Pin J. P., Mouillac B., Durroux T. (2010). Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol. 6, 587–59410.1038/nchembio.396 - DOI - PMC - PubMed
    1. Ali S., Drucker D. J. (2009). Benefits and limitations of reducing glucagon action for the treatment of type 2 diabetes. Am. J. Physiol. Endocrinol. Metab. 296, E415–E42110.1152/ajpendo.90887.2008 - DOI - PubMed
    1. Angers S., Salahpour A., Joly E., Hilairet S., Chelsky D., Dennis M., Bouvier M. (2000). Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U.S.A. 97, 3684–368910.1073/pnas.060590697 - DOI - PMC - PubMed
    1. Authier F., Desbuquois B. (2008). Glucagon receptors. Cell. Mol. Life Sci. 65, 1880–189910.1007/s00018-008-7479-6 - DOI - PMC - PubMed

LinkOut - more resources

  NODES
Association 1
twitter 2