Abstract
The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adrenergic beta-Agonists / chemistry
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Adrenergic beta-Agonists / metabolism
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Adrenergic beta-Antagonists / chemistry
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Adrenergic beta-Antagonists / metabolism
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Amino Acid Sequence
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Bacteriophage T4 / enzymology
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Binding Sites
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Cell Line
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Cell Membrane / chemistry
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Cell Membrane / metabolism
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Crystallization
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Crystallography, X-Ray
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Drug Inverse Agonism
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Humans
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Immunoglobulin Fab Fragments / chemistry
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Immunoglobulin Fab Fragments / metabolism
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Ligands
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Models, Molecular
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Molecular Sequence Data
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Muramidase / chemistry
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Muramidase / metabolism
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Propanolamines / chemistry
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Propanolamines / metabolism
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Protein Conformation
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Receptors, Adrenergic, beta-2 / chemistry*
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Receptors, Adrenergic, beta-2 / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
Substances
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Adrenergic beta-Agonists
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Adrenergic beta-Antagonists
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Immunoglobulin Fab Fragments
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Ligands
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Propanolamines
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Receptors, Adrenergic, beta-2
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Recombinant Fusion Proteins
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carazolol
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Muramidase