The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and <sup>1</sup>H NMR spectroscopy in aqueous and membrane‐like environments

Choi, Giltsu; Landin, Judith S.; Xie, Xiang‐Qun

doi:10.1034/j.1399-3011.2002.21012.x

Cited by 23 publications

(20 citation statements)

References 36 publications

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“…Our NMR results reveal that the C-terminal juxtamembrane domains of both CB 1 and CB 2 acquire helical conformations in membrane mimetic DPC micelles. These findings are consistent with the results of circular dichroism measurements of the peptides (15,19).…”

supporting

confidence: 82%

“…Studies have been carried out to understand the three-dimensional structure of the CB receptors and their mechanisms of action by using computer molecular modeling and NMR approaches (11)(12)(13)(14)(15)(16)(17). Bramblett et al (11) first analyzed the secondary structure of the CB 1 receptor based upon calculated hydrophobicity and variability profiles to predict the regions of ␣-helicity and then predicted the three-dimensional structure model of the CB 1 receptor showing an extracellular N-terminal, seven transmembrane helical segments, three intracellular loops, and three extracellular loops, as well as a juxtamembrane helical C-terminal domain.…”

mentioning

confidence: 99%

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NMR Structural Comparison of the Cytoplasmic Juxtamembrane Domains of G-protein-coupled CB1 and CB2 Receptors in Membrane Mimetic Dodecylphosphocholine Micelles

Xie

Chen

2005

Journal of Biological Chemistry

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The fourth cytoplasmic domain, the so-called C-terminal juxtamembrane segment or helix VIII, has been identified in numerous G-protein-coupled receptors and exhibits unique functional characteristics. Efforts have been devoted to studying the juxtamembrane segment in order to understand the biological importance of the segment in G-protein activation of the cannabinoid CB 1 and CB 2 receptors. Recent biochemical data revealed that the CB 1 C-terminal juxtamembrane peptide fragment CB 1 -(401-417) can directly activate the G-protein and also showed that the specificity of the signal transduction activation by the C-terminal juxtamembrane region is unique to the CB 1 receptor but not to the CB 2 receptor (Mukhopadhyay, S., and Howlett, A. C. (2001) Eur. J. Biochem. 268, 499 -505). However, there is experimental work, not yet reported, on the conformational analyses and structural comparison between the respective helix VIII segments of the two receptors. In the present study, we have examined the conformational specificities of the cytoplasmic helical domains for both cannabinoid receptors. Three-dimensional structural features of two synthetic CB 1 and CB 2 peptides, CB 1 I397-G418 and CB 2 I298-K319, respectively, in membrane mimetic DPC micelles were studied using a combined high resolution NMR and computer modeling approach. Comparisons of the NMR-determined structures of the two peptides as well as their correspondent mutant peptides revealed their conformational properties and salt bridge dissimilarity, which might help us to understand the different structural roles of the fourth cytoplasmic helices in the function and regulation of CB 1 and CB 2 receptors.The cannabinoid (CB) 1 receptor subtypes, CB 1 and CB 2, have been cloned and classified into the class A rhodopsin-like family of the seven transmembrane G-protein-coupled receptors (GPCRs) (1, 2). These subtypes have also been identified as important drug discovery targets for numerous potential therapeutic applications, including antiemetics, appetite stimulants, analgesics, glaucoma treatment, and immune suppression (3, 4). The CB 1 receptor is located in the central and peripheral nervous system (1, 5, 6), whereas the CB 2 receptor is distributed peripherally in non-neuronal tissues, particularly in immune cells (2, 6). The CB 1 receptor interacts with the pertussis toxin-sensitive G i/o family of G-proteins to inhibit adenylate cyclase (7) and to regulate N-type Ca 2ϩ channels and inwardly rectifying K ϩ channels in the central nervous system (8 -10). The CB 2 receptor is expressed in high quantities in the human spleen and tonsils and exhibits 44% amino acid identity to the CB 1 receptor throughout the whole protein. The CB2 receptor is likely involved in the signal transduction processes in the immune system (6).Studies have been carried out to understand the three-dimensional structure of the CB receptors and their mechanisms of action by using computer molecular modeling and NMR approaches (11)(12)(13)(14)(15)(16)(17). Bramblett et al. (11) fir...

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confidence: 82%

mentioning

confidence: 99%

NMR Structural Comparison of the Cytoplasmic Juxtamembrane Domains of G-protein-coupled CB1 and CB2 Receptors in Membrane Mimetic Dodecylphosphocholine Micelles

Xie

Chen

2005

Journal of Biological Chemistry

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“…NMR in conjunction with CD spectroscopy confirmed the presence of an H8 for the CB 1 andCB 2 receptors, the Ste2p receptor, the β 1 and β 2 receptors, the angiotensin AT 1A receptor, and the bradykinin B 2 receptor in detergent [70–75]. In Fig.…”

Section: Determination Of the 3d Structure Of Gpcrsmentioning

confidence: 92%

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

Tikhonova¹,

Costanzi²

2009

CPD

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G protein-coupled receptors (GPCRs) are a large superfamily of signaling proteins expressed on the plasma membrane. They are involved in a wide range of physiological processes and, therefore, are exploited as drug targets in a multitude of therapeutic areas. In this extent, knowledge of structural and functional properties of GPCRs may greatly facilitate rational design of modulator compounds. Solution and solid-state nuclear magnetic resonance (NMR) spectroscopy represents a powerful method to gather atomistic insights into protein structure and dynamics. In spite of the difficulties inherent the solution of the structure of membrane proteins through NMR, these methods have been successfully applied, sometimes in combination with molecular modeling, to the determination of the structure of GPCR fragments, the mapping of receptor-ligand interactions, and the study of the conformational changes associated with the activation of the receptors. In this review, we provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures. KeywordsG protein-coupled receptors (GPCRs); NMR; three-dimensional structures; ligand recognition; receptor activation G protein-coupled receptors (GPCRs), also known as seven transmembrane-spanning receptors (7TMRs), are a large superfamily of signaling proteins expressed on the plasma membrane that function as receivers for extracellular stimuli [1]. About 1000 GPCRs have been identified in the human genome [2]. Since their signaling is involved in numerous physiological functions and pathological conditions, GPCRs constitute the molecular target of a significant percentage of the currently marketed drugs. Moreover, many additional members of the superfamily have been identified as potential targets for the treatment of a variety of diseases and are the object of substantial drug discovery efforts. From the molecular point of view, all GPCRs share a common molecular architecture, being composed of a single polypeptide chain folded into a bundle of seven α-helical transmembrane domains (TMs) connected by three extracellular and three intracellular loops (ELs and ILs). The N-terminus is located in the extracellular space, while the C-terminus is in the cytosol (Fig. (1)).Given that structure-based drug discovery is an efficient method to rationally design novel drugs and improve the properties of old drugs, the scientific community has been striving for a long time to shed light onto the elusive structure-function relationships of GPCRs employing a variety of direct biophysical and indirect biochemical methods [3]. The most direct method that has been used is X-ray crystallography. However, up until now, this technique has been *address for correspondence: stefanoc@mail.nih.gov. NIH Public Access Author ManuscriptCurr Pharm Des. Author manuscript; available in PMC 2010 January 5. Published in final edited form as:Curr Pharm Des. 2009 ; 15(35): 4003-4016. NIH-PA Author ManuscriptNIH-PA Author Manuscript...

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“…The amphipathic cavity consists of a hydrophilic and a hydrophobic centre framed by polar or aromatic residues, respectively (see Table 2). In addition, the amphipathic cytoplasmic helix 8 was identified by NMR experiments [72] and computer modeling. Recently, they reported to have refined the model by preparing recombinant transmembrane helix proteins, reconstituting them into helical structures in solution, and finally incorporating these helix structures into the CB2 model [73].…”

Section: Tm7 and C-terminusmentioning

confidence: 99%

Targeting the Cannabinoid CB2 Receptor: Mutations, Modeling and Development of CB2 Selective Ligands

Raitio¹,

Salo²,

Nevalainen

et al. 2005

CMC

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Since the discovery of the cannabinoid CB2 receptor in 1993, there has been a growing interest to clarify the importance of this G-protein coupled receptor (GPCR) for human physiology, and to investigate it as a possible target for current and future drug development. Several mutation studies have examined the receptor activation and structure of the receptor binding cavity. Additionally, 3D models for the CB2 receptor have been constructed to aid in perceiving important ligand-receptor interactions. In recent years, many research groups have succeeded in synthesizing new CB2 selective ligands. This review focuses on (i) important features for ligand recognition and/or receptor activation at CB2, derived from mutation and modeling studies, and (ii) recent advances in the field of CB2 selective ligands.

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The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and ¹H NMR spectroscopy in aqueous and membrane‐like environments

Cited by 23 publications

References 36 publications

NMR Structural Comparison of the Cytoplasmic Juxtamembrane Domains of G-protein-coupled CB1 and CB2 Receptors in Membrane Mimetic Dodecylphosphocholine Micelles

NMR Structural Comparison of the Cytoplasmic Juxtamembrane Domains of G-protein-coupled CB1 and CB2 Receptors in Membrane Mimetic Dodecylphosphocholine Micelles

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

Targeting the Cannabinoid CB2 Receptor: Mutations, Modeling and Development of CB2 Selective Ligands

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The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and 1H NMR spectroscopy in aqueous and membrane‐like environments

Cited by 23 publications

References 36 publications

NMR Structural Comparison of the Cytoplasmic Juxtamembrane Domains of G-protein-coupled CB1 and CB2 Receptors in Membrane Mimetic Dodecylphosphocholine Micelles

NMR Structural Comparison of the Cytoplasmic Juxtamembrane Domains of G-protein-coupled CB1 and CB2 Receptors in Membrane Mimetic Dodecylphosphocholine Micelles

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

Targeting the Cannabinoid CB2 Receptor: Mutations, Modeling and Development of CB2 Selective Ligands

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The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and ¹H NMR spectroscopy in aqueous and membrane‐like environments