期刊
EUROPEAN JOURNAL OF PHARMACOLOGY
卷 630, 期 1-3, 页码 10-18出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ejphar.2009.12.022
关键词
CRTH2; G protein-coupled receptor; Internalization; GRKs; PKC; Arrestin
资金
- Natural Sciences and Engineering Research Council of Canada
- Fonds de la Recherche en Sante du Quebec
- Canadian Institutes of Health Research
The molecular mechanisms regulating the trafficking of the CRTH2 receptor are poorly understood In the present study, we characterize C-terminal tail determinants involved in the agonist-induced trafficking of the CRTH2 receptor for prostaglandin D-2 Our results showed that progressive deletion of C-terminal tail residues from amino acid 395 up to 337 gradually impaired CRTH2 internalization by similar to 50% as measured by ELISA in HEK293 cells. Surprisingly, further deletion of the C-tail to amino acid 328 or 317 resulted in receptor mutants displaying internalization similar to the wild-type receptor. Individual mutations of Asp(330), Ser(331), Glu(332), and Leu(333) to Ala in the C-tail of the full length receptor resulted in a 45% increase in internalization of the receptor mutants relative to the wild-type receptor Pretreatment with the recycling inhibitor monensin increased internalization of the wild-type receptor but did not affect that of the D330A, S331A, E332A and L333A mutants, indicating that these residues are part of a recycling motif Further experiments revealed that Asp(330), Ser(331) and Glu(332) are not only involved in receptor recycling, but are also required for promotion of CRTH2 internalization by GRK2 and GRK5 Site-directed mutagenesis identified Thr(347) as a major site for PKC-induced internalization of the receptor. Confocal microscopy revealed that arrestin-3 dissociated from the receptor after agonist stimulation and internalization, suggesting that CRTH2 is a class A G protein-coupled receptor Our study identified specific amino acids in the CRTH2 receptor C-tail implicated in the agonist-induced internalization and the recycling of the receptor (C) 2009 Elsevier B V All rights reserved
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