Carboxypeptidase M is a positive allosteric modulator of the kinin B1 receptor

Xianming Zhang, Fulong Tan, Randal A. Skidgel*

*Corresponding author for this work

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Ligand binding to extracellular domains of Gprotein-coupled receptors can result in novel and nuanced allosteric effects on receptor signaling. We previously showed that the protein-protein interaction of carboxypeptidase M (CPM) and kinin B1 receptor (B1R) enhances B1R signaling in two ways; 1) kinin binding to CPM causes a conformational activation of the B1R, and 2) CPM-generated des-Arg-kinin agonist is efficiently delivered to the B1R. Here, we show CPM is also a positive allosteric modulator of B1R signaling to its agonist, des-Arg10-kallidin (DAKD). In HEK cells stably transfected with B1R, co-expression of CPM enhanced DAKD-stimulated increases in intracellular Ca 2+ or phosphoinositide turnover by a leftward shift of the doseresponse curve without changing the maximum. CPM increased B1R affinity for DAKD by ∼5-fold but had no effect on basal B1R-dependent phosphoinositide turnover. Soluble, recombinant CPM bound to HEK cells expressing B1Rs without stimulating receptor signaling. CPM positive allosteric action was independent of enzyme activity but depended on interaction of its C-terminal domain with the B1R extracellular loop 2. Disruption of the CPM/B1R interaction or knockdown of CPM in cytokine-treated primaryhumanendothelial cells inhibited the allostericenhancement of CPM on B1R DAKD binding or ERK1/2 activation. CPM also enhanced the DAKD-induced B1R conformational change as detected by increased intramolecular fluorescence or bioluminescence resonance energy transfer. Thus, CPM binding to extracellular loop 2 of the B1R results in positive allosteric modulation of B1R signaling, and disruption of this interaction could provide a novel therapeutic approach to reduce pathological B1R signaling.

Original languageEnglish (US)
Pages (from-to)33226-33240
Number of pages15
JournalJournal of Biological Chemistry
Volume288
Issue number46
DOIs
StatePublished - Nov 15 2013

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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