Peptides and proteins with a C-terminal basic amino acid (e.g., bradykinin, anaphylatoxins) are potent mediators involved in may pathological, inflammatory processes. Removal of the C-terminal basic amino acid byu a carboxypeptidase can inactivated or alter the activity of this type of mediator. This proposal focuses on the newly described carboxypeptidase (CP)-M, which is plasma- membrane bound in many cells and tissues and cleaves C-terminal basic amino acids. The enzyme definitely differs from plasma CP- N or CP-H which is in intracellular secretory granules. The study of CP-M will fill a gap in our knowledge of how basic peptides and proteins can be processed or catabolized when they gain access to sites that are inaccessible to CP-N (i.e, extravascular) or CP-H (i.e., extrascellular). Our long-term objective is to understand how CP-M controls peptide activity under physiological and pathological conditions.
The specific aims are: 1. Purify CP-M and raise antiserum. 2. Compare the enzymatic, physical and immunological characteristic of CP-M to those of other B-type carboxypeptidase. 3. Determine the N-terminal sequence of CP-M and the sequences of some internal peptides. 4. Isolate and sequence a cDNA clone corresponding to CP-M in order to deduce the protein sequence. 5. From the primary sequence of CP-M determine: the signal or activation peptide, if present, potential membrane binding region(s), glycosylation sites and possible active site residues. 6. Determine the mode of attachment of CP-M to the cell membrane. 7. Determine the localization of CP-M in kidney, placenta and cultured cells by immuno-electron microscopy. 8. Investigate the enzymatic activity of CP-M on the cell membrane by comparing kinetics of hydrolysis of biologically active peptides by soluble CP-M vs.membrane-bound CP-M. 9. Investigate the substrate specificity of CP-M with a series of synthetic peptides. 10. Study the synthesis, membrane attachment and membrane polarity, postranslational processing and possible mechanisms of release in cultured kidney cells. Accomplishing these objectives will provide new information on the localization and biochemical structural, and enzymatic characteristics of CP-M. It is hoped that the results will be applicable to the study of the control of peptide activity in normal or pathological situations.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Illinois at Chicago
Schools of Medicine
United States
Zip Code
Zhang, Xianming; Brovkovych, Viktor; Zhang, Yongkang et al. (2015) Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling. Cell Signal 27:90-103
Zhang, Xianming; Tan, Fulong; Brovkovych, Viktor et al. (2013) Carboxypeptidase M augments kinin B1 receptor signaling by conformational crosstalk and enhances endothelial nitric oxide output. Biol Chem 394:335-45
Zhang, Xianming; Lowry, Jessica L; Brovkovych, Viktor et al. (2012) Characterization of dual agonists for kinin B1 and B2 receptors and their biased activation of B2 receptors. Cell Signal 24:1619-31
Brovkovych, Viktor; Zhang, Yongkang; Brovkovych, Svitlana et al. (2011) A novel pathway for receptor-mediated post-translational activation of inducible nitric oxide synthase. J Cell Mol Med 15:258-69
Zhang, Xianming; Tan, Fulong; Brovkovych, Viktor et al. (2011) Cross-talk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling. J Biol Chem 286:18547-61
Erdös, Ervin G; Tan, Fulong; Skidgel, Randal A (2010) Angiotensin I-converting enzyme inhibitors are allosteric enhancers of kinin B1 and B2 receptor function. Hypertension 55:214-20
Kuhr, F; Lowry, J; Zhang, Y et al. (2010) Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors. Neuropeptides 44:145-54
Kuhr, Frank K; Zhang, Yongkang; Brovkovych, Viktor et al. (2010) Beta-arrestin 2 is required for B1 receptor-dependent post-translational activation of inducible nitric oxide synthase. FASEB J 24:2475-83