The major determinant of exendin-4/glucagon-like peptide 1 differential affinity at the rat glucagon-like peptide 1 receptor N-terminal domain is a hydrogen bond from SER-32 of exendin-4

BACKGROUND AND PURPOSE

Exendin-4 (exenatide, Ex4) is a high-affinity peptide agonist at the glucagon-like peptide-1 receptor (GLP-1R), which has been approved as a treatment for type 2 diabetes. Part of the drug/hormone binding site was described in the crystal structures of both GLP-1 and Ex4 bound to the isolated N-terminal domain (NTD) of GLP-1R. However, these structures do not account for the large difference in affinity between GLP-1 and Ex4 at this isolated domain, or for the published role of the C-terminal extension of Ex4. Our aim was to clarify the pharmacology of GLP-1R in the context of these new structural data.

EXPERIMENTAL APPROACH

The affinities of GLP-1, Ex4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Molecular dynamics coupled with in silico mutagenesis were used to model and interpret the data.

KEY RESULTS

The membrane-tethered NTD of hGLP-1R displayed similar affinity for GLP-1 and Ex4 in sharp contrast to previous studies using the soluble isolated domain. The selectivity at rGLP-1R for Ex4(9–39) over Ex4(9–30) was due to Ser-32 in the ligand. While this selectivity was not observed at hGLP-1R, it was regained when Glu-68 of hGLP-1R was mutated to Asp.

CONCLUSIONS AND IMPLICATIONS

GLP-1 and Ex4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of Ex4 and Asp-68 of rGLP-1R, which is not formed with Glu-68 of hGLP-1R, is responsible for the improved affinity of Ex4 at the rat receptor.     

A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation

P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG₁) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M_r) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIbα). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of ¹²⁵I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825±2886, mean ±SD) (K_d=1.5±0.5 nm ) compared to resting platelets (2801±1278, mean ±SD) (K_d=1.5±0.6 nm ), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG₁, F(ab′)₂ or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf ) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell–cell contact, may play an active role in platelet–platelet interactions.     

A near-fatal reaction during granulocyte transfusion of a neonate

Although reactions to granulocyte transfusions in neonates are rarely reported, we observed a near-fatal pulmonary reaction, presumably due to white cell antibodies, in a neonate with Rh hemolytic disease. The hemolytic disease was being treated with exchange transfusions, and at 2 days after the infant's birth, bacterial sepsis was suspected and granulocyte transfusions were begun. The first granulocyte transfusion (Day 3) was uneventful. Five minutes after the beginning of the second granulocyte transfusion (Day 4), severe respiratory distress, hypotension, bradycardia, cyanosis, and acidosis suddenly occurred. The infant's serum obtained after the reaction contained granulocytotoxic and B-lymphocytotoxic antibodies that reacted with leukocytes from the second granulocyte donor. Antibodies could not be detected either in the initial infant serum or in maternal serum. However, an antileukocyte antibody was present in the serum of a parous woman donor. We used plasma from this woman to prepare reconstituted whole blood for the exchange transfusion that we performed immediately preceding the second granulocyte transfusion. Despite the sequence of events, an irrefutable cause-and-effect mechanism could not be established because the properties of the donor and neonatal antibodies were similar, but not identical. However, this catastrophic event emphasizes both the potential for adverse effects of granulocyte transfusions in neonates and the need for caution when transfusing blood from parous women.     

Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell-cell adhesion molecule that is expressed on circulating platelets, on leukocytes, and at the intercellular junctions of vascular endothelial cells and mediates the interactions of these cells during the process of transendothelial cell migration. The cDNA for PECAM-1 encodes an open reading frame of 738 amino acids (aa) that is organized into a 27- aa signal peptide, a 574-aa extracellular domain composed of 6 Ig homology units, and a relatively long cytoplasmic tail of 118 aa containing multiple sites for posttranslational modification and postreceptor signal transduction. To provide a molecular basis for the precise evaluation of the structure and function of this transmembrane glycoprotein, we have determined the organization of the human PECAM-1 gene. The PECAM-1 gene, which has been localized to human chromosome 17, is a single-copy gene of approximately 65 kb in length and is broken into 16 exons by introns ranging in size from 86 to greater than 12,000 bp in length. Typical of other members of the Ig superfamily, each of the extracellular Ig homology domains is encoded by a separate exon, consistent with PECAM-1 having arisen by gene duplication and exon shuffling of ancestral Ig superfamily genes. However, the cytoplasmic domain was found to be surprisingly complex, being encoded by seven short exons that may represent discrete functional entities. Alternative splicing of the cytoplasmic tail appears to generate multiple PECAM-1 isoforms that may regulate phosphorylation, cytoskeletal association, and affinity modulation of the mature protein. Finally, a processed pseudogene having 76% identity with PECAM- 1 cDNA was identified and localized to human chromosome 3. These findings should have important implications for structure/function analysis of PECAM-1 and its role in vascular adhesive interactions.     

The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils

The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface- bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor- alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.     

The canonical NF-kappaB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion

The role of mammary epithelial cell (MEC) NF-κB in tumor progression in vivo is unknown, as murine NF-κB components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-κB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-κB, the importance of MEC NF-κB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-κB (IκBαSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-κB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-κB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that IκBαSR inhibited tumor stem cell pathways. IκBαSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-κB contributes to mammary tumorigenesis. As we show that NF-κB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-κB-dependent mammary tumorigenesis.