Importance of lipid-exposed residues in transmembrane segment four for family B calcitonin receptor homo-dimerization

Harikumar, Kaleeckal G.; Ball, Alicja M.; Sexton, Patrick M.; Miller, Laurence J.

doi:10.1016/j.regpep.2010.06.001

Cited by 26 publications

(39 citation statements)

References 34 publications

(61 reference statements)

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“…S4) abolished the BRET signal ( Fig. 1 A and B), consistent with TM4 constituting the principal dimer interface for GLP-1Rs as well as other family B GPCRs (17,18,31). Disruption of GLP-1R dimerization either by coincubation of GLP-1R BRET constructs with GLP-1R TM4 (Fig.…”

Section: Glp-1 Receptor Forms Functionally Important Homodimerssupporting

confidence: 72%

“…As previously observed with other family B GPCRs (17,18,25), analysis of receptor-receptor interaction in live cells using a combination of static and saturation bioluminescence resonance energy transfer (BRET) analyses revealed constitutive, specific homooligomerization of the GLP-1R (Fig.…”

Section: Glp-1 Receptor Forms Functionally Important Homodimerssupporting

confidence: 64%

“…As seen with the related calcitonin and secretin family B GPCRs (17,18), disruption of the TM4 interface by either GLP-1R TM4 peptides or by mutation of the hydrophobic face of TM4 (L256A, V259A, or G252A, L256A, V259A; Fig. S4) abolished the BRET signal ( Fig.…”

Section: Glp-1 Receptor Forms Functionally Important Homodimersmentioning

confidence: 71%

“…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).…”

mentioning

confidence: 99%

“…There is also emerging evidence for functionally significant heterodimerization (25)(26)(27). Furthermore, although there is an emerging theme in which dimerization contributes to high-affinity peptide binding and cAMP signaling (17,18), how dimerization contributes more globally to receptor signaling and whether it plays a role in ligand-directed stimulus bias are unknown.…”

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confidence: 99%

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

Harikumar

Wootten

Pinon

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The glucagon-like peptide-1 receptor (GLP-1R) is a family B G proteincoupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent highaffinity binding to GLP-1(7-36)NH 2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.allosteric modulation | biased signaling | G protein-coupled receptors | family B GPCRs G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface proteins and play crucial roles in virtually every physiological process. Their widespread abundance, yet selective distribution, and ability to couple to a variety of signaling and effector systems make them extremely attractive targets for drug development (1). Recently, there has been increasing interest in the stoichiometry of receptors involved in GPCR signaling complexes and how this may impact on receptor function and drug discovery (2-4).With the exception of family C GPCRs, where obligate dimerization can occur (4), the role of oligomerization in GPCR function has remained controversial (2-8), and this has been the subject of a number of recent reviews (9-11). Although there is an increasing body of evidence supporting dimerization of GPCRs as a widespread feature of GPCR biology, including numerous studies on family A GPCRs, whether these are stable, transient, constitutive, or ligand dependent, and how they impact on receptor function and drug discovery are less clear, and general rules for oligomeric behavior are not evident (9-16). Even where effects on signaling are studied, these are generally linked to a single pathway and the role of dimerization in the control of receptor engagement and preference for distinct intracellular signaling intermediates (i.e., signal bias) is virtually unstudied.For family B GPCRs, which encompass many ther...

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Section: Glp-1 Receptor Forms Functionally Important Homodimerssupporting

confidence: 72%

Section: Glp-1 Receptor Forms Functionally Important Homodimerssupporting

confidence: 64%

Section: Glp-1 Receptor Forms Functionally Important Homodimersmentioning

confidence: 71%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Harikumar

Wootten

Pinon

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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A Novel Approach to Quantify G-Protein-Coupled Receptor Dimerization Equilibrium Using Bioluminescence Resonance Energy Transfer

Kufareva

Stephens

Gilliland

et al. 2013

Methods in Molecular Biology

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Along with other resonance energy transfer techniques, bioluminescence resonance energy transfer (BRET) has emerged as an important method for demonstrating protein–protein interactions in cells. In the field of G protein-coupled receptors, including chemokine receptors, BRET has been widely used to investigate homo- and heterodimerization, a feature of their interactions that is emerging as integral to function and regulation. While demonstrating the existence of dimers for a given receptor proved to be fairly straightforward, quantitative comparisons of different receptors or mutants are nontrivial because of inevitable variations in the expression of receptor constructs. The uncontrollable parameters of the cellular expression machinery make amounts of transfected DNA extremely poor predictors for the expression levels of BRET donor and acceptor receptor constructs, even in relative terms. In this chapter, we show that properly accounting for receptor expression levels is critical for quantitative interpretation of BRET data. We also provide a comprehensive account of expected responses in all types of BRET experiments and propose a framework for uniform and accurate quantitative treatment of these responses. The framework allows analysis of both homodimer and heterodimer BRET data. The important caveats and obstacles for quantitative treatment are outlined, and the utility of the approach is illustrated by its application to the homodimerization of wild-type (WT) and mutant forms of the chemokine receptor CXCR4.

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Secretin Receptor Dimerization. Prototypic of Class B GPCR Behavior

Harikumar

Miller

2017

G-Protein-Coupled Receptor Dimers

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Importance of lipid-exposed residues in transmembrane segment four for family B calcitonin receptor homo-dimerization

Cited by 26 publications

References 34 publications

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

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

A Novel Approach to Quantify G-Protein-Coupled Receptor Dimerization Equilibrium Using Bioluminescence Resonance Energy Transfer

Secretin Receptor Dimerization. Prototypic of Class B GPCR Behavior

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