Paola Bianchi Guimarães1, Rafael Filippelli da Silva1, Carolina Caldas Hoff2, Liliam Fernandes3, Clovis Ryuichi Nakaie1, Jair Ribeiro Chagas1, Adriana Karaoglanovic Carmona1, Michael Bader4, João Bosco Pesquero5. 1. Departamento de Biofísica, Universidade Federal de São Paulo, Rua Pedro de Toledo 669, 9° andar fundos, São Paulo, SP, 04039-032, Brazil. 2. Departamento de Biociências, Universidade Federal de São Paulo, Santos, SP, Brazil. 3. Instituto de Ciências Ambientais, Químicas e Farmacêuticas (ICAQF), Universidade Federal de São Paulo, Diadema, SP, Brazil. 4. Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. 5. Departamento de Biofísica, Universidade Federal de São Paulo, Rua Pedro de Toledo 669, 9° andar fundos, São Paulo, SP, 04039-032, Brazil. jbpesquero@unifesp.br.
Abstract
INTRODUCTION: Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. AIMS: We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. METHODS: Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 -/- ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B1R, were also used. RESULTS: CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. CONCLUSIONS: Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.
INTRODUCTION:Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. AIMS: We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. METHODS: Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 -/- ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B1R, were also used. RESULTS:CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. CONCLUSIONS: Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.
Entities:
Keywords:
B1 receptor; Carboxypeptidase M; Enzymatic activity; Kinins; Microdomains
Authors: Vanessa F Merino; Mihail Todiras; Luciana A Campos; Vera Saul; Elena Popova; Ovidiu C Baltatu; João B Pesquero; Michael Bader Journal: J Mol Med (Berl) Date: 2008-04-19 Impact factor: 4.599
Authors: L R Steranka; D C Manning; C J DeHaas; J W Ferkany; S A Borosky; J R Connor; R J Vavrek; J M Stewart; S H Snyder Journal: Proc Natl Acad Sci U S A Date: 1988-05 Impact factor: 11.205
Authors: Gabriel R Estrela; Frederick Wasinski; Marcos F Gregnani; Leandro C Freitas-Lima; Adriano C Arruda; Rafael Leite Morais; Denise Mac Malheiros; Niels O S Camara; João Bosco Pesquero; Michael Bader; Carlos Castilho Barros; Ronaldo Carvalho Araújo Journal: Front Mol Biosci Date: 2020-05-20