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Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

ABSTRACT: The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied ?(2)-adrenoceptor (?(2)AR) in complex with the heterotrimeric G protein Gs (G?s??). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the ?-helical domain of G?s appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the ?-helical domain on the Ras-like domain of G?s. Molecular modeling of the ?-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of G?s-GTP?S. These data argue that the ?-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

SUBMITTER: Westfield GH

PROVIDER: S-EPMC3179071 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

Westfield Gerwin H GH Rasmussen Søren G F SG Su Min M Dutta Somnath S DeVree Brian T BT Chung Ka Young KY Calinski Diane D Velez-Ruiz Gisselle G Oleskie Austin N AN Pardon Els E Chae Pil Seok PS Liu Tong T Li Sheng S Woods Virgil L VL Steyaert Jan J Kobilka Brian K BK Sunahara Roger K RK Skiniotis Georgios G

Proceedings of the National Academy of Sciences of the United States of America 20110913 38

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β(2)-adrenoceptor (β(2)AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architect ...[more]

PMID: 21914848

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Project description:1 The principal aim of the present study was to determine whether long-term treatment of human lung mast cells (HLMC) with the clinically-relevant beta(2)-adrenoceptor agonists, salbutamol and terbutaline, leads to desensitization of beta(2)-adrenoceptor-mediated responses in these cells. 2 The non-selective beta-adrenoceptor agonist, isoprenaline, and the selective beta(2)-adrenoceptor agonists, salbutamol and terbutaline, inhibited the IgE-mediated release of histamine from HLMC. Salbutamol (pD(2); 7.7+/-0.3) and terbutaline (pD(2); 7.3+/-0.2) were roughly equipotent as inhibitors of histamine release although both agonists were less potent than isoprenaline (pD(2); 8.6+/-0.2). 3 Isoprenaline (10(-5) M), salbutamol (10(-5) M) and terbutaline (10(-5) M) enhanced total cell cAMP levels in HLMC over basal by 361+/-90, 150+/-38 and 165+/-35%, respectively. 4 Long-term exposure (24 h) of HLMC to either salbutamol (10(-7) M) or terbutaline (10(-7) M) led to a subsequent reduction in the effectiveness of salbutamol and terbutaline (both 10(-9)-10(-4) M) to inhibit histamine release. However, salbutamol was significantly (P<0.05) more effective than terbutaline at promoting the functional desensitization. 5 Radioligand binding studies, using iodinated cyanopindolol, were performed to determine beta(2)-adrenoceptor density in cell membranes after pretreatment (24 h) of cells with either salbutamol (10(-6) M) or terbutaline (10(-6) M). Both agonists reduced beta(2)-adrenoceptor density in membranes to about the same extent (approximately 25% reduction) but these changes in receptor density were not statistically significant (P>0.05). 6 These data indicate that long-term exposure of mast cells to salbutamol causes greater levels of desensitization to beta(2)-adrenoceptor-mediated responses in HLMC than terbutaline. These findings may have wider clinical significance in the context of asthma treatment as compromised mast cell inhibition could result following long-term exposure of mast cells to short-acting bronchodilators.

Dataset Information

Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

Publications

Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

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