title: Neutrophil thrombospondin receptors are linked to GTP-binding proteins.
abstract: The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP-binding proteins (G-proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP-mediated motility. Both PT and CT inhibited TSP-mediated chemotaxis and substrate-associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 microM) nor 8-bromo-cAMP (300 microM) significantly affected TSP-mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 +/- 0.3 to 14.2 +/- 0.1 pmol/10(6) cells). Although we could not test the PT sensitivity of TSP priming for FMLP-mediated chemotaxis (as PT inhibits FMLP-mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP-dependent priming of FMLP-mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G-proteins was obtained by examining the effect of TSP on alpha-subunit ADP-ribosylation, GTPase activity, and GTP gamma S binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT-dependent ribosylation of Ci alpha 2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G-proteins. However, TSP did not block the CT-dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of Gi alpha 2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 +/- 2.7% of control values). In addition, GTP gamma S binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 +/- 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G-proteins, a subpopulation of which also associates with FMLP receptors.
authors.affiliation: Department of Pediatrics, University of Michigan, Ann Arbor 48109, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Suchard
authors.forename: P J
authors.initials: PJ
authors.lastname: Mansfield
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D018955:CD36 Antigens / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D002772:Cholera Toxin / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000502:physiology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D037342:Pertussis Toxin; D011485:Protein Binding; D015398:Signal Transduction; D019699:Thrombospondins; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D018955:CD36 Antigens; D008562:Membrane Glycoproteins; D019699:Thrombospondins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D002772:Cholera Toxin; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D006152:Cyclic GMP