Identification of a Novel Site within G Protein α Subunits Important for Specificity of Receptor-G Protein Interaction

Heydorn, Arne; Ward, Richard J.; Jørgensen, Rasmus; Rosenkilde, Mette M.; Frimurer, Thomas M.; Milligan, Graeme; Kostenis, Evi

doi:10.1124/mol.66.2.250

Cited by 45 publications

(49 citation statements)

References 44 publications

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“…The importance of the two bridges for chemokine-induced activation of CCR8 was then investigated using an IP 3 accumulation assay. In this assay, the G␣ i signal from CCR8 was converted into a G␣ q response by co-transfecting the cells with the chimeric G protein G␣ ⌬6qi4myr (32). This allows measurements of the G␣ i signaling of CCR8 (G␣ i inhibits adenylate cyclase, and the G␣ i activity is measured as inhibition of forskolin-induced cAMP production) into a G␣ q signal (G␣ q activates phospholipase C, and the activity is measured as IP 3 accumulation) without interference with the receptor structure but only by interference with the G protein downstream of the receptor.…”

Section: Resultsmentioning

confidence: 99%

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Barington

Rummel

Lückmann

et al. 2016

Journal of Biological Chemistry

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Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr 184 (Cys ؉ 1) and Tyr 187 (Cys ؉ 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr 187 in TMIV (Phe 171 ) and TMV (Trp 194 ). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr 187 . This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.Chemokines (chemotactic cytokines) regulate the differentiation, activation, and recruitment of leukocytes. They also play important roles in several physiological mechanisms outside the immune system such as organogenesis and angiogenesis (1, 2). With ϳ50 members, these cytokines exert their effects through chemokine receptors (23 members), which belong to class A of the family of seven-transmembrane (7TM) 2 G protein-coupled receptors (3). The implications of the chemokine system in a vast number of human diseases (3) have increased the interest in developing potent, selective, and clinically useful chemokine receptor antagonists.The binding of a chemokine to its cognate receptor is initially driven by electrostatic interactions between the overall positively charged chemokine and the negatively charged extracellular surface of the receptor. Then interactions between the chemokine N terminus and residues in the main binding pocket of the receptor trigger receptor activation (4 -6). In contrast, small molecule ligands bind deeper in the main binding pocket and constrain the receptors in either active or inactive conformations (7,8). Whereas most mapping studies of small molecules have focused on the transmembrane areas, newer studies as well as crystal structures of class A receptors suggest that extracellular receptor regions, in particular ex...

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Section: Resultsmentioning

confidence: 99%

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Barington

Rummel

Lückmann

et al. 2016

Journal of Biological Chemistry

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“…The most extensive analysis of this interaction involves family A GPCRs (Kostenis et al, 1997;Heydorn et al, 2004). Much less is currently known about the G protein-binding site within family B GPCRs.…”

Section: Discussionmentioning

confidence: 99%

Functional Importance of a Structurally Distinct Homodimeric Complex of the Family B G Protein-Coupled Secretin Receptor

Gao¹,

Harikumar²,

Dong³

et al. 2009

Mol Pharmacol

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Oligomerization of G protein-coupled receptors has been described, but its structural basis and functional importance have been inconsistent. Here, we demonstrate that the agonist occupied wild-type secretin receptor is predominantly in a guanine nucleotide-sensitive high-affinity state and exhibits negative cooperativity, whereas the monomeric receptor is primarily in a guanine nucleotide-insensitive lower affinity state. We previously demonstrated constitutive homodimerization of this receptor through the lipid-exposed face of transmembrane (TM) IV. We now use cysteine-scanning mutagenesis of 14 TM IV residues, bioluminescence resonance energy transfer (BRET), and functional analysis to map spatial approximations and functional importance of specific residues in this complex. All, except for three helix-facing mutants, trafficked to the cell surface, where secretin was shown to bind and elicit cAMP production. Cells expressing complementary-tagged receptors were treated with cuprous phenanthroline to establish disulfide bonds between spatially approximated cysteines. BRET was measured as an indication of receptor oligomerization and was repeated after competitive disruption of oligomers with TM IV peptide to distinguish covalent from noncovalent associations. Although all constructs generated a significant BRET signal, this was disrupted by peptide in all except for single-site mutants replacing five residues with cysteine. Of these, covalent stabilization of receptor homodimers through positions of Gly 243 , Ile 247 , and Ala 250 resulted in a GTP-sensitive highaffinity state of the receptor, whereas the same procedure with Ala 246 and Phe 240 mutants resulted in a GTP-insensitive lower affinity state. We propose the existence of a functionally important, structurally specific high-affinity dimeric state of the secretin receptor, which may be typical of family B G proteincoupled receptors.

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“…Construction of G␣ q and G␣ qG66X (X ϭ D, N, V, K) in the pcDNA1.1 expression vector was reported previously (Heydorn et al, 2004). The C-terminally modified G␣ q subunits G␣ qi5/s5 were created by introducing, in a two-piece ligation, a polymerase chain reaction-based 175-base pair BglII-NsiI fragment containing the desired codon changes.…”

Section: Dna Constructionmentioning

confidence: 99%

“…On the level of the G protein, both G␣ and the ␤␥ subunit complexes are likely to possess receptor contact sites (Conklin et al, 1993;Kisselev et al, 1995;Taylor et al, 1996). Within the G␣ subunit, several key regions governing GPCR-G protein coupling specificity have been identified so far; these include 1) the extreme C terminus (Conklin et al, 1993;Kostenis et al, 1997a;Liu et al, 2002;Havlickova et al, 2003), 2) the extreme N terminus (Kostenis et al, 1997b;, 3) a region between ␣4 and ␣5 helices (Bae et al, 1997;Cai et al, 2001;, and 4) a region within the loop linking the N-terminal ␣-helix to the ␤1 strand of the Ras-like domain (Blahos et al, 2001) and the linker I region connecting the ras like with the helical domain (Heydorn et al, 2004). Whereas all of these domains have been evaluated independently for their specific interaction with GPCRs, relatively little is known about whether these regions interact in an additive or cooperative manner.…”

mentioning

confidence: 99%

“…Whereas all of these domains have been evaluated independently for their specific interaction with GPCRs, relatively little is known about whether these regions interact in an additive or cooperative manner. We have previously reported that a glycine residue within linker I (glycine 66 in G␣ q ), which is highly conserved among G␣ subunits across all species, is a key residue for constraining the fidelity of G protein recognition by ligand-activated GPCRs (Heydorn et al, 2004). Molecular modeling studies suggested that glycine 66 is located in a key position within linker I and is the only amino acid that can be accommodated in this location without interfering with the proper arrangement of both helical and GTPase domains of G␣.…”

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confidence: 99%

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A Highly Conserved Glycine within Linker I and the Extreme C Terminus of G Protein α Subunits Interact Cooperatively in Switching G Protein-Coupled Receptor-to-Effector Specificity

Kostenis¹,

Martini

Ellis

et al. 2004

J Pharmacol Exp Ther

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Numerous studies have attested to the importance of the extreme C terminus of G protein ␣ subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor recognition by G␣ q proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant G␣ q proteins in which glycine was replaced with various amino acids and the C-terminal five G␣ q residues with the corresponding G␣ i or G␣ s sequence. Coupling properties of the mutated G␣ q proteins were determined after coexpression with a panel of 13 G i -and G s -selective receptors and compared with those of G␣ proteins modified in only one module. G␣ proteins modified in both modules are significantly more efficacious in channeling non-G q -selective receptors to G q -mediated signaling events compared with those containing each module alone. Additive effects of both modules were observed even if individual modules lacked an effect on GPCR-to-effector specificity. Dually modified G␣ proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves cooperative interactions between the extreme C terminus and linker I of G␣ proteins and that distinct determinants of selectivity exist for individual receptors.Accurate signal transduction demands that seven transmembrane G protein-coupled receptors (GPCRs) selectively regulate a few of the many available and closely related G proteins expressed within a given cell. Conversely, G proteins must distinguish between selected groups of GPCRs, and each member of the four different subfamilies of G␣ proteins (G␣ i/o , G␣ s , G␣ q , and G␣ 12/13 ) is capable of interacting with multiple GPCRs. Understanding the structural requirements that determine the exquisite selectivity of receptor-G protein recognition will clarify how specificity is maintained in living cells. Importantly, it may also suggest ways to manipulate signal transduction, pharmacologically or genetically, or simply enable development of assays that allow receptor activation to be linked to a signal transduction event of choice.

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Identification of a Novel Site within G Protein α Subunits Important for Specificity of Receptor-G Protein Interaction

Cited by 45 publications

References 44 publications

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Functional Importance of a Structurally Distinct Homodimeric Complex of the Family B G Protein-Coupled Secretin Receptor

A Highly Conserved Glycine within Linker I and the Extreme C Terminus of G Protein α Subunits Interact Cooperatively in Switching G Protein-Coupled Receptor-to-Effector Specificity

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