Conformational Landscape and Dynamics of G Protein-Coupled Receptors

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15 pages, 4904 KiB

Open AccessArticle

Dexmedetomidine-Induced Aortic Contraction Involves Transactivation of the Epidermal Growth Factor Receptor in Rats

by Soo Hee Lee, Seong-Chun Kwon, Seong-Ho Ok, Seung Hyun Ahn, Sung Il Bae, Ji-Yoon Kim, Yeran Hwang, Kyeong-Eon Park, Mingu Kim and Ju-Tae Sohn

Int. J. Mol. Sci. 2022, 23(8), 4320; https://doi.org/10.3390/ijms23084320 - 13 Apr 2022

Cited by 2 | Viewed by 1420

Abstract

In this study, we examined whether aortic contraction, induced by the alpha-2 adrenoceptor agonist dexmedetomidine, is involved in the transactivation of the epidermal growth factor receptor (EGFR) in isolated endothelium-denuded rat aortas. Additionally, we aimed to elucidate the associated underlying cellular mechanisms. The effects of the alpha-2 adrenoceptor inhibitor rauwolscine, EGFR tyrosine kinase inhibitor AG1478, Src kinase inhibitors PP1 and PP2, and matrix metalloproteinase inhibitor GM6001 on EGFR tyrosine phosphorylation and c-Jun NH₂-terminal kinase (JNK) phosphorylation induced by dexmedetomidine in rat aortic smooth muscles were examined. In addition, the effects of these inhibitors on dexmedetomidine-induced contraction in isolated endothelium-denuded rat aorta were examined. Dexmedetomidine-induced contraction was inhibited by the alpha-1 adrenoceptor inhibitor prazosin, rauwolscine, AG1478, PP1, PP2, and GM6001 alone or by a combined treatment with prazosin and AG1478. AG1478 (3 × 10⁻⁶ M) inhibited dexmedetomidine-induced contraction in isolated endothelium-denuded rat aortas pretreated with rauwolscine. Dexmedetomidine-induced EGFR tyrosine and JNK phosphorylation were inhibited by rauwolscine, PP1, PP2, GM6001, and AG1478. Furthermore, dexmedetomidine-induced JNK phosphorylation reduced upon EGFR siRNA treatment. Therefore, these results suggested that the transactivation of EGFR associated with dexmedetomidine-induced contraction, mediated by the alpha-2 adrenoceptor, Src kinase, and matrix metalloproteinase, caused JNK phosphorylation and increased calcium levels. Full article

(This article belongs to the Topic Conformational Landscape and Dynamics of G Protein-Coupled Receptors)

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19 pages, 5273 KiB

Open AccessArticle

Universal Properties and Specificities of the β₂-Adrenergic Receptor-G_s Protein Complex Activation Mechanism Revealed by All-Atom Molecular Dynamics Simulations

by Argha Mitra, Arijit Sarkar and Attila Borics

Int. J. Mol. Sci. 2021, 22(19), 10423; https://doi.org/10.3390/ijms221910423 - 27 Sep 2021

Cited by 3 | Viewed by 3014

Abstract

G protein-coupled receptors (GPCRs) are transmembrane proteins of high pharmacological relevance. It has been proposed that their activity is linked to structurally distinct, dynamically interconverting functional states and the process of activation relies on an interconnecting network of conformational switches in the transmembrane domain. However, it is yet to be uncovered how ligands with different extents of functional effect exert their actions. According to our recent hypothesis, based on indirect observations and the literature data, the transmission of the external stimulus to the intracellular surface is accompanied by the shift of macroscopic polarization in the transmembrane domain, furnished by concerted movements of highly conserved polar motifs and the rearrangement of polar species. In this follow-up study, we have examined the β₂-adrenergic receptor (β₂AR) to see if our hypothesis drawn from an extensive study of the μ-opioid receptor (MOP) is fundamental and directly transferable to other class A GPCRs. We have found that there are some general similarities between the two receptors, in agreement with previous studies, and there are some receptor-specific differences that could be associated with different signaling pathways. Full article

(This article belongs to the Topic Conformational Landscape and Dynamics of G Protein-Coupled Receptors)

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15 pages, 2749 KiB

Open AccessArticle

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

by Vanessa Boritzki, Harald Hübner, Anni Allikalt, Peter Gmeiner and Birgitta M. Wöhrl

Int. J. Mol. Sci. 2021, 22(16), 8647; https://doi.org/10.3390/ijms22168647 - 11 Aug 2021

Viewed by 2485

Abstract

The human dopamine receptors D_2S and D₃ belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D_2S and D₃ receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate K_D-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies. Full article

(This article belongs to the Topic Conformational Landscape and Dynamics of G Protein-Coupled Receptors)

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