NK1 receptor fused to β-arrestin displays a single-component, high-affinity molecular phenotype

被引:30
作者
Martini, L
Hastrup, H
Holst, B
Fraile-Ramos, A
Marsh, M
Schwartz, TW
机构
[1] Univ Copenhagen, Panum Inst, Dept Pharmacol, Mol Pharmacol Lab, DK-2200 Copenhagen, Denmark
[2] 7TM Pharma AS, Copenhagen, Denmark
[3] UCL, Dept Biochem, London, England
[4] UCL, Cell Biol Unit, MRC, Mol Cell Biol Lab, London, England
关键词
D O I
10.1124/mol.62.1.30
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G(11) and Gs pathways. The NK1-beta-arrestin1 fusion construct bound non-peptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.
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页码:30 / 37
页数:8
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共 34 条
[31]   Interaction with β-arrestin determines the difference in internalization behavior between β1- and β2-adrenergic receptors [J].
Shiina, T ;
Kawasaki, A ;
Nagao, T ;
Kurose, H .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (37) :29082-29090
[32]   Endocytosis and recycling of the HIV coreceptor CCR5 [J].
Signoret, N ;
Pelchen-Matthews, A ;
Mack, M ;
Proudfoot, AEI ;
Marsh, M .
JOURNAL OF CELL BIOLOGY, 2000, 151 (06) :1281-1293
[33]   BINDING OF PURIFIED RECOMBINANT BETA-ARRESTIN TO GUANINE-NUCLEOTIDE-BINDING-PROTEIN-COUPLED RECEPTORS [J].
SOHLEMANN, P ;
HEKMAN, M ;
PUZICHA, M ;
BUCHEN, C ;
LOHSE, MJ .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1995, 232 (02) :464-472
[34]   Cellular trafficking of G protein-coupled receptor/β-arrestin endocytic complexes [J].
Zhang, J ;
Barak, LS ;
Anborgh, PH ;
Laporte, SA ;
Caron, MG ;
Ferguson, SSG .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (16) :10999-11006