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.