Stable expression of the human kinin B1 receptor in chinese hamster ovary cells: Characterization of ligand binding and effector pathways

Caroline E. Austin, Alexander Faussner, Helen E. Robinson, Sarvajit Chakravarty, Donald J. Kyle, Joan M. Bathon, David Proud

Research output: Contribution to journalArticlepeer-review

96 Scopus citations

Abstract

To delineate ligand binding and functional characteristics of the human B1 kinin receptor, a stable clone of Chinese hamster ovary cells expressing a single class of binding sites for [3H]des-Arg10-lysylbradykinin with a K(d) of 0.3 nM and a B(max) of 38 fmol/mg protein (~40,000 receptors/cell) was isolated. Studies with peptide analogs showed that a lysine residue at position 1 (based on the lysylbradykinin sequence) of ligands was essential for high affinity binding to the human B1 receptor. In marked contrast to cloned Chinese hamster ovary cells expressing the human kinin B2 receptor, which internalized approximately 80% of the ligand within 5 min upon exposure to 2 nM [3H]bradykinin, exposure of cells expressing the B1 receptor to 1 nM [3H]des-Argl10-lysylbradykinin resulted in minimal ligand internalization. Stimulation of the B1 receptor led to inositol phosphate generation and transient increases in intracellular calcium, confirming coupling to phospholipase C, while immunoprecipitation of photoaffinity- labeled G-proteins from membranes indicated specific coupling of the receptor to Gαq/11 and Gαi1,2. The B1, unlike the B2, receptor does not desensitize (as demonstrated by continuous phosphoinositide hydrolysis), enhancing the potential role of this receptor during inflammatory events.

Original languageEnglish (US)
Pages (from-to)11420-11425
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number17
DOIs
StatePublished - Apr 25 1997

ASJC Scopus subject areas

  • Biochemistry

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