Role of the Conserved DRY Motif on G Protein Activation of Rat Angiotensin II Receptor Type 1A

doi:10.1006/bbrc.2002.6670

Biochemical and Biophysical Research Communications

Volume 292, Issue 2, 29 March 2002, Pages 362-367

https://doi.org/10.1006/bbrc.2002.6670 Get rights and content

Abstract

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp–Arg–Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT_1A) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP₃ production, and the acidification of the medium in response to Ang II were determined. The effects of GTPγS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP₃ production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP₃ and acidify medium comparable with wild type AT_1A. These results indicate as follows; Asp¹²⁵ is essential for intracellular signal transduction involving G protein coupling, Arg¹²⁶ is essential for coupling of G_q protein but not other G proteins, and Tyr¹²⁷ is not important for G protein coupling.

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Identification of distinct conformations of the angiotensin-II type 1 receptor associated with the G<inf>q/11</inf> protein pathway and the β-arrestin pathway using molecular dynamics simulations
2015, Journal of Biological Chemistry
Citation Excerpt :
We also monitored H-bonds formed between D1253.39 and R1263.50 and between residues of the DRY motif and other neighboring residues, but no interaction involving the DRY motif of AT1 was linked to the opening of the G protein-binding site. This is in line with the alternative role proposed to be played by these residues for the AT1 receptor and other GPCRs (56–59). Indeed, they have been proposed to be rather involved in the direct interaction with the G protein rather than modulating the conformational stability of the receptor.
Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT₁) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the G_q/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT₁ receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT₁ receptor, the N111G-AT₁ receptor (constitutively active and biased for the G_q/11 pathway), and the D74N-AT₁ receptor (biased for the β-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT₁, N111G-AT₁, and AngII-N111G-AT₁ receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT₁ receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT₁ receptor. The biased agonist [Sar¹,Ile⁸]AngII also showed a preference for the ground state of the WT-AT₁ receptor compared with AngII. These results suggest that activation of the G_q/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the β-arrestin pathway is linked to the stabilization of the ground state of the receptor.
The N111G and D74N mutations bias the AT₁ receptor for the G_q/11 and β-arrestin pathways, respectively.
Structural rearrangements of theAT₁ receptor are induced by the N111G mutation and AngII.
Activation of the G_q/11 and β-arrestin pathways is associated with a decreased and increased stability, respectively, of the ground state of the receptor.
Distinct conformations of AT₁ receptor are associated with distinct pathways.
Characterization of G protein coupling mediated by the conserved D1343.49 of DRY motif, M2416.34, and F2516.44 residues on human CXCR1
2015, FEBS Open Bio
Citation Excerpt :
Substitution mutations of Asp1343.49 to asparagine (D134N) or valine (D134V) resulted in nonfunctional receptors that were devoid of ligand binding. Similar findings have been reported in TM 3.49 of GPCRs such as rat angiotensin II (Ang II) receptor type 1A (AT1A) [44], vasopressin V1a receptor (V1aR) [45], muscarinic receptor [46], and cannabinoid 2 (CB2R) [47,48]. Although D134 mutants are expressed on the surface of transfected cells, they barely bind to the ligand IL-8, suggesting that inactivity of D134 mutants could be due to impaired receptor folding.
CXCR1, a receptor for interleukin-8 (IL-8), plays an important role in defending against pathogen invasion during neutrophil-mediated innate immune response. Human CXCR1 is a G protein-coupled receptor (GPCR) with its characteristic seven transmembrane domains (TMs). Functional and structural analyses of several GPCRs have revealed that conserved residues on TM3 (including the highly conserved Asp-Arg-Tyr (DRY) motif) and TM6 near intracellular loops contain domains critical for G protein coupling as well as GPCR activation. The objective of this study was to elucidate the role of critical amino acid residues on TM3 near intracellular loop 2 (i2) and TM6 near intracellular loop 3 (i3), including S132^3.47 (Baldwin location), D134^3.49, M241^6.34, and F251^6.44, in G protein coupling and CXCR1 activation. The results demonstrate that mutations of D134^3.49 at DRY motif of CXCR1 (D134N and D134V) completely abolished the ligand binding and functional response of the receptor. Additionally, point mutations at positions 241 and 251 between TM6 and i3 loop generated mutant receptors with modest constitutive activity via Gα15 signaling activation. Our results show that D134^3.49 on the highly conserved DRY motif has a distinct role for CXCR1 compared to its homologues (CXCR2 and KSHV-GPCR) in G protein coupling and receptor activation. In addition, M241^6.34 and F251^6.44 along with our previously identified V247^6.40 on TM6 are spatially located in a “hot spot” likely essential for CXCR1 activation. Identification of these amino acid residues may be useful for elucidating mechanism of CXCR1 activation and designing specific antagonists for the treatment of CXCR1-mediated diseases.
A type 1 cholecystokinin receptor mutant that mimics the dysfunction observed for wild type receptor in a high cholesterol environment
2014, Journal of Biological Chemistry
Citation Excerpt :
It is less well conserved than the other residues, often replaced with a cysteine, histidine, or valine. Mutation of this residue has often had no, or minimal, functional impact (29–33). In this study, we have focused on the Y140A mutant of the CCK1R, now establishing a stable mutant receptor-bearing cell line and expanding its functional characterization.
Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A G protein-coupled receptor-specific (E/D)RY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced the importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu³⁵⁶ target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.
Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol
2012, Journal of Lipid Research
Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.
Molecular classification of an elasmobranch angiotensin receptor: Quantification of angiotensin receptor and natriuretic peptide receptor mRNAs in saltwater and freshwater populations of the Atlantic stingray
2010, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Citation Excerpt :
Four extracellular cysteines involved in the formation of two disulfide bonds are conserved across all taxa (Nishimura, 2001). Also conserved in the D. sabina AT protein are a DRY motif (aa 125–127) involved in G-protein coupling (Ohyama et al., 2002), an NPxxY motif (aa 298–302, where x is any amino acid) required for signal transduction (Laporte et al., 1996), and a serine/threonine-rich region (aa 326–338) involved in receptor desensitization and internalization (Smith et al., 1998; Kule et al., 2004). Several residues critical to ligand–receptor interaction and subsequent activation of mammalian AT1 are also present in D. sabina: D74, N111, K199, W253, D281 and Y292 (Groblewski et al., 1997; Le et al., 2002).
Among the most conserved osmoregulatory hormone systems in vertebrates are the renin–angiotensin system (RAS) and the natriuretic peptides (NPs). We examined the RAS and NP system in the euryhaline Atlantic stingray, Dasyatis sabina (Lesueur). To determine the relative sensitivity of target organs to these hormonal systems, we isolated cDNA sequences encoding the D. sabina angiotensin receptor (AT) and natriuretic peptide type-B receptor (NPR-B). We then determined the tissue-specific expression of their mRNAs in saltwater D. sabina from local Texas waters and an isolated freshwater population in Lake Monroe, Florida. AT mRNA was most abundant in interrenal tissue from both populations. NPR-B mRNA was most abundant in rectal gland tissue from both populations, and also highly abundant in the kidney of saltwater D. sabina. This study is the first to report the sequence of an elasmobranch angiotensin receptor, and phylogenetic analysis indicates that the D. sabina receptor is more similar to AT₁ vs. AT₂ proteins. This classification is further supported by molecular analysis of AT₁ and AT₂ proteins demonstrating conservation of AT₁-specific amino acid residues and motifs in D. sabina AT. Molecular classification of the elasmobranch angiotensin receptor as an AT₁-like protein provides fundamental insight into the evolution of the vertebrate RAS.
Molecular determinants of angiotensin II type 1 receptor functional selectivity
2009, Journal of Molecular and Cellular Cardiology
The angiotensin AT₁ receptor is an important pharmacological target in the treatment of cardiovascular disorders, such as hypertension, diabetic nephropathy, cardiac hypertrophy, arrhythmia and failure. Simultaneously, the AT₁ receptor has emerged to be a prominent model for the emerging concept that receptors may attain multiple active states with differentiated functional outcomes. Two major signalling pathways are employed by the AT₁ receptor, namely 1) the canonical G_q protein-dependent activation of inositol phosphate turnover and intracellular calcium release, and 2) G protein-independent recruitment of β-arrestin-scaffolded signalling complexes that activate protein kinase pathways. Different states of receptor activation with preference for individual downstream pathways (functional selectivity) have been demonstrated in mutational studies of the AT₁ receptor and by pharmacological probing with analogues of angiotensin II. These studies also provide clues about the conformational changes that underlie different functional outcomes. In this review, we evaluate current knowledge of the molecular determinants of AT₁ receptor activation, which may distinguish G protein-dependent and -independent behaviour. While G protein activation is known to be detrimental, G protein-independent signalling by the AT₁ receptor has been associated with phenotypes such as cell survival and renewal, regulation of cardiac contraction and cell migration. It is therefore currently hypothesized that selective blockade of G protein actions and simultaneous activation of G protein-independent signalling will prove to be a feasible strategy for improved cardiovascular therapy. The pharmacological perspectives of functional selectivity by receptors, such as the AT₁ receptor, urge the elucidation of molecular mechanisms that govern disparate signalling events.

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¹: To whom correspondence should be addressed at Department of Clinical Nursing and Pediatrics, Yamanashi Medical University, 1110, Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, Japan. Fax: +81-55-273-6605. E-mail: [email protected].

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Biochemical and Biophysical Research Communications

Abstract

References (29)

Domains for G-protein coupling in angiotensin II receptor type I: Studies by site-directed mutagenesis

Biochem. Biophys. Res. Commun.

A domain for G protein coupling in carboxyl-terminal tail of rat angiotensin II receptor type 1A

J. Biol. Chem.

Constitutive activation of A(3) adenosine receptors by site-directed mutagenesis

Biochem. Biophys. Res. Commun.

Role of the highly conserved Asp–Arg–Tyr motif in signal transduction of the CB2 cannabinoid receptor

FEBS Lett.

Identification and characterization of a new binding site for angiotensin II in mouse neuroblastoma neuro-2A cells

Biochem. Biophys. Res. Commun.

Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor

Nature

Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor

Nature

Site-directed mutagenesis of human beta-adrenergic receptors: Substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase

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

Mutation of a highly conserved aspartic acid in the beta 2 adrenergic receptor: Constitutive activation, structual instability, and conformational rearrangement of transmembrane segment 6

Mol. Pharmacol.

Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: Conserved aspartic acids play important roles in receptor function

Mol. Pharmacol.

Site-directed mutagenesis of alpha 2A-adrenergic receptors: Identification of amino acids involved in ligand binding and receptor activation by agonists

Mol. Pharmacol.

Mutation of the DRY motif that preserve beta 2-adrenoceptor coupling

Receptors Channels

Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization

Mol. Endocrinol.

An arginine residue conserved in most G protein-coupled receptors is essential for the function of the m1 muscarinic receptor

Mol. Pharmacol.

Cited by (32)

Identification of distinct conformations of the angiotensin-II type 1 receptor associated with the G<inf>q/11</inf> protein pathway and the β-arrestin pathway using molecular dynamics simulations

Characterization of G protein coupling mediated by the conserved D134<sup>3.49</sup> of DRY motif, M241<sup>6.34</sup>, and F251<sup>6.44</sup> residues on human CXCR1

A type 1 cholecystokinin receptor mutant that mimics the dysfunction observed for wild type receptor in a high cholesterol environment

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

Molecular classification of an elasmobranch angiotensin receptor: Quantification of angiotensin receptor and natriuretic peptide receptor mRNAs in saltwater and freshwater populations of the Atlantic stingray

Molecular determinants of angiotensin II type 1 receptor functional selectivity

Regular ArticleRole of the Conserved DRY Motif on G Protein Activation of Rat Angiotensin II Receptor Type 1A

Abstract

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor

Nature

Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor

Nature

Site-directed mutagenesis of human beta-adrenergic receptors: Substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase

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

Mutation of a highly conserved aspartic acid in the beta 2 adrenergic receptor: Constitutive activation, structual instability, and conformational rearrangement of transmembrane segment 6

Mol. Pharmacol.

Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: Conserved aspartic acids play important roles in receptor function

Mol. Pharmacol.

Site-directed mutagenesis of alpha 2A-adrenergic receptors: Identification of amino acids involved in ligand binding and receptor activation by agonists

Mol. Pharmacol.

Mutation of the DRY motif that preserve beta 2-adrenoceptor coupling

Receptors Channels

Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization

Mol. Endocrinol.

An arginine residue conserved in most G protein-coupled receptors is essential for the function of the m1 muscarinic receptor

Mol. Pharmacol.

Regular Article
Role of the Conserved DRY Motif on G Protein Activation of Rat Angiotensin II Receptor Type 1A