Stephen C.-T. Lam

Identification of a Novel Integrin α6β1 Binding Site in the Angiogenic Inducer CCN1 (CYR61)

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including α6β1. Previous studies have shown that CCN1 interaction with integrin α6β1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin α6β1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-α6 and anti-β1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating α6β1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other α6β1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits α6β1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin α6β1 directly, we perform affinity chromatography and show that integrin α6β1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin α6β1, providing the basis for the development of peptide mimetics to examine the functional role of α6β1 in angiogenesis.

Identification of a Novel Integrin αvβ3 Binding Site in CCN1 (CYR61) Critical for Pro-angiogenic Activities in Vascular Endothelial Cells

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin αvβ3 to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin αvβ3. Immobilized synthetic V2 peptide supports αvβ3-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin αvβ3 and abolished αvβ3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified αvβ3 and impaired in αvβ3-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds αvβ3 and supports αvβ3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary αvβ3 binding site in soluble CCN1, whereas additional cryptic αvβ3 binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin αvβ3 and provide a new approach for dissecting αvβ3-specific CCN1 functions both in cultured cells and in the organism.

Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61) SELECTIVE INACTIVATION OF INTEGRIN α6β1-HEPARAN SULFATE PROTEOGLYCAN CORECEPTOR-MEDIATED CELLULAR FUNCTIONS

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin α6β1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel α6β1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support α6β1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with α6β1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished α6β1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain αvβ3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in α6β1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for α6β1-HSPG-dependent CCN1 activities and provide a strategy to investigate these α6β1-HSPG-specific activities in vivo.

The P2Y12 antagonists, 2-methylthioadenosine 5'-monophosphate triethylammonium salt and cangrelor (ARC69931MX), can inhibit human platelet aggregation through a Gi-independent increase in cAMP levels.

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors.

Anti-peptide antibodies against the human blood platelet thromboxane A2/prostaglandin H2 receptor: Production, purification and characterization

Two anti-peptide antibodies have been raised against the human blood platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. Based on the published sequence of the placental TXA2/PGH2 receptor, two decapeptide segments were selected as potential antigens: one in the first extracellular loop corresponding to residue 89 through 98, and the other in the C-terminal region of the intracellular domain corresponding to residue 314 through 323. Rabbits were immunized with each peptide, and the antisera were subjected to a two-step purification procedure. The IgG fraction was purified using a DEAE Affi-Gel Blue column, and the peptide-specific IgG was further purified by affinity chromatography employing each peptide as the immobilized ligand. The combined purification factor for both procedures was approximately 60-fold. By ELISA, both antibodies displayed immunoreactivity toward their synthetic antigens, solubilized platelet membranes and affinity-purified TXA2/PGH2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA2/PGH2 receptor protein (55 kDa); and (2) each antibody recognized a single band (55 kDa) in solubilized platelet membranes. These findings establish antibody specificity for the human platelet TXA2/PGH2 receptor protein. Functional analysis demonstrated that neither antibody interfered with ADP- or U46619-induced platelet aggregation of [3H]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epitopes are separate from the TXA2/PGH2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human platelet TXA2/PGH2 receptor. These antibodies should prove to be of value in the further investigation of the platelet TXA2/PGH2 receptor.