tailieunhanh - Báo cáo khoa học: Alanine screening of the intracellular loops of the human bradykinin B2 receptor – effects on receptor maintenance, G protein activation and internalization

The bradykinin B2 receptor is coupled to G protein Gq⁄ 11 and becomes sequestered into intracellular compartments after activation. To more clo-sely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. | ỊFEBS Journal Alanine screening of the intracellular loops of the human bradykinin B2 receptor - effects on receptor maintenance G protein activation and internalization Alexander Faussner1 Goeran Wennerberg1 Steffen Schiissler1 Jens Feierler1 Cornelia Seidl1 Marianne Jochum1 and David Proud2 1 Ludwig-Maximilians-Universitat Munchen Abteilung fur Klinische Chemie und Klinische Biochemie Muenchen Germany 2 Department of Physiology and Biophysics University of Calgary Alberta Canada Keywords affinity shift B9430 G protein-coupled receptor icatibant semi-active conformation Correspondence A. Faussner Ludwig-Maximilians-Universitaet Muenchen Abteilung Klinische Chemie und Klinische Biochemie Nussbaumstrasse 20 D-80336 Muenchen Germany Fax 49 89 5160 4740 Tel 49 89 5160 2602 E-mail . Received 27 January 2009 revised 9 April 2009 accepted 22 April 2009 doi The bradykinin B2 receptor is coupled to G protein Gq 11 and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions we systematically mutated all three intracellular loops ICLs either as point mutations or in groups of three to five amino acids to Ala obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and with the exception of triple mutant DRY fi AAA retained the ability to specifically bind 3H bradykinin. The binding affinities at 4 or 37 C of several mutants differed considerably from those determined for the wild-type receptor indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or 3H bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies whereas two mutations in ICL-3 resulted in

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