The central and peripheral cannabinoid receptors (CB1 and CB2): Structural characterization and active site elucidation using covalent probes

Activation of different classes of plasma membrane receptors regulates the activity of practically every cell of the body. The vast majority of these receptors belong to the super family of G protein coupled receptors (GPCRs). Human CB1 and CB2, like other GPCRs, have a similar integral membrane topology. The transmembrane domains (TMs) are predicted to be those regions that are involved in ligand-receptor interaction. Although the two subtypes share affinity for a few cannabinergic ligands and areas that are important for ligand binding and receptor subtype specificity have been identified, the actual ligand-binding sites remain unknown and we cannot assume ligands bind in the same manner to both receptor subtypes because of the non-conserved amino acid sequence and their contrasting physiological roles. Hence, determination of the ligand receptor-binding motifs will facilitate the design of receptor-selective, bioactive, and safe synthetic cannabinoids. ^ In order to do this we need proof that CB1 and CB2 bind the same ligands differently. This can be achieved by using high affinity covalent ligands that have similar affinity towards both the CB1 and CB2 receptors and identifying key amino acid residues in the two receptors that interact with the covalent moieties of these ligands. This approach has been termed as Ligand Based Structural Biology by our lab. ^ With the peripheral Cannabinoid Receptor CB2, biochemical techniques were used to identify the ligand binding motif. This involved a number of technically-demanding experimental steps like functional over-expression of CB2 cannabinoid receptors in insect cells using the baculovirus system, interaction of covalent cannabinergic ligands with CB2 receptor preparations, purification of the ligand-receptor covalent complex using affinity chromatography and identification of the amino acid residue(s) through mass spectroscopic analysis of the digested fragments. Using this method, the binding motifs of AM1336 (CB2 antagonist) and AM841 (CB2 agonist) have been characterized. ^ The central cannabinoid receptor (CB1), is more difficult to purify, so site-directed mutagenesis and subsequent biochemical testing were used to identify the ligand binding motifs. In order to do this a global set of cysteine substitutions (cysteine to serine) in the full length and truncated human CB1 receptors was generated and saturation binding assays were performed to assess the expression levels. CB1 selective covalent probes were used to label the receptor and binding assays with [ 3H] CP55940 were undertaken to assess the extent to which, if any, the mutations would alter the covalent binding ability of the cannabinergic ligands. Using this methodology, the cysteine in the transmembrane helix 6 (C6.47(355)) has been identified as the site of covalent attachment of AM3677, an anandamide analog and the cysteine in transmembrane helix 7 (C7.42(386)) as the site of covalent attachment of AM4073, a classical cannabinoid. ^ In conclusion, the work presented here demonstrates for the first time a complete peptide mapping of a GPCR beyond rhodopsin using nanoLC coupled to the 4000 Q-Trap MS and the high accuracy mass measurement LTQ-FT MS and a MALDI-TOF. Also, the key binding motifs for different classes of cannabinergic ligands with respect to both the central and peripheral cannabinoid receptors have been identified and unequivocally proved. And this work has laid the foundation for future experiments to characterize the binding site and ultimately produce sufficient amounts of purified protein for crystallization and NMR studies.

The central and peripheral cannabinoid receptors (CB1 and CB2): Structural characterization and active site elucidation using covalent probes

Activation of different classes of plasma membrane receptors regulates the activity of practically every cell of the body. The vast majority of these receptors belong to the super family of G protein coupled receptors (GPCRs). Human CB1 and CB2, like other GPCRs, have a similar integral membrane topology. The transmembrane domains (TMs) are predicted to be those regions that are involved in ligand-receptor interaction. Although the two subtypes share affinity for a few cannabinergic ligands and areas that are important for ligand binding and receptor subtype specificity have been identified, the actual ligand-binding sites remain unknown and we cannot assume ligands bind in the same manner to both receptor subtypes because of the non-conserved amino acid sequence and their contrasting physiological roles. Hence, determination of the ligand receptor-binding motifs will facilitate the design of receptor-selective, bioactive, and safe synthetic cannabinoids. ^ In order to do this we need proof that CB1 and CB2 bind the same ligands differently. This can be achieved by using high affinity covalent ligands that have similar affinity towards both the CB1 and CB2 receptors and identifying key amino acid residues in the two receptors that interact with the covalent moieties of these ligands. This approach has been termed as Ligand Based Structural Biology by our lab. ^ With the peripheral Cannabinoid Receptor CB2, biochemical techniques were used to identify the ligand binding motif. This involved a number of technically-demanding experimental steps like functional over-expression of CB2 cannabinoid receptors in insect cells using the baculovirus system, interaction of covalent cannabinergic ligands with CB2 receptor preparations, purification of the ligand-receptor covalent complex using affinity chromatography and identification of the amino acid residue(s) through mass spectroscopic analysis of the digested fragments. Using this method, the binding motifs of AM1336 (CB2 antagonist) and AM841 (CB2 agonist) have been characterized. ^ The central cannabinoid receptor (CB1), is more difficult to purify, so site-directed mutagenesis and subsequent biochemical testing were used to identify the ligand binding motifs. In order to do this a global set of cysteine substitutions (cysteine to serine) in the full length and truncated human CB1 receptors was generated and saturation binding assays were performed to assess the expression levels. CB1 selective covalent probes were used to label the receptor and binding assays with [ 3H] CP55940 were undertaken to assess the extent to which, if any, the mutations would alter the covalent binding ability of the cannabinergic ligands. Using this methodology, the cysteine in the transmembrane helix 6 (C6.47(355)) has been identified as the site of covalent attachment of AM3677, an anandamide analog and the cysteine in transmembrane helix 7 (C7.42(386)) as the site of covalent attachment of AM4073, a classical cannabinoid. ^ In conclusion, the work presented here demonstrates for the first time a complete peptide mapping of a GPCR beyond rhodopsin using nanoLC coupled to the 4000 Q-Trap MS and the high accuracy mass measurement LTQ-FT MS and a MALDI-TOF. Also, the key binding motifs for different classes of cannabinergic ligands with respect to both the central and peripheral cannabinoid receptors have been identified and unequivocally proved. And this work has laid the foundation for future experiments to characterize the binding site and ultimately produce sufficient amounts of purified protein for crystallization and NMR studies.

Reduction of Proteinuria through Podocyte Alkalinization

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pH(i)). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pH(i)

CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1–dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease