Epidermal Growth Factor-like Repeats of Thrombospondins Activate Phospholipase Cγ and Increase Epithelial Cell Migration through Indirect Epidermal Growth Factor Receptor Activation

Abstract

Thrombospondin (TSP) 1 is a trimeric multidomain protein that contains motifs that recognize distinct host cell receptors coupled to multiple signaling pathways. Selected TSP1-induced cellular responses are tyrosine kinasedependent, and TSP1 contains epidermal growth factor (EGF)-like repeats. Specific receptor interactions or functions for the EGF-like repeats have not been identified. We asked whether one or more biological responses to TSP1 might be explained through EGF receptor (EGFR) activation. In A431 cells, TSP1 increased autophosphorylation of Tyr-1068 of EGFR in a dose-and time-dependent manner. The ability of TSP1 to activate EGFR was replicated by the tandem EGF-like repeats as a recombinant protein. The three EGFlike repeats alone produced a high level of Tyr-1068 phosphorylation. EGF-like repeats from TSP2 and TSP4 also activated EGFR. Tyr-1068 phosphorylation was less when individual EGF-like repeats were tested or flanking sequences were added to the three EGF-like repeats. TSP1 and its EGF-like repeats also increased phosphorylation of EGFR Tyr-845, Tyr-992, Tyr-1045, Tyr-1086, and Tyr-1173, activated phospholipase C␥, and increased cell migration. No evidence was found for binding of the EGF-like repeats to EGFR. Instead, EGFR activation in response to TSP1 or its EGF-like repeats required matrix metalloprotease activity, including activity of matrix metalloprotease 9. Access to the ligand-binding portion of the EGFR ectodomain was also required. These findings suggest release of an endogenous EGFR ligand in response to ligation of a second unknown receptor by the TSPs.

NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling

“…7, B, C, and E, and 8, C-I). To be catalytically active, NEU1 must operate within a multiprotein complex comprising NEU1, cathepsin A, and ␤-galactosidase (19,20,29). In those experiments where NEU1 alone was overexpressed, it is possible that our results are understated.…”

“…EGFR Is an in Vivo NEU1 Substrate-EGFR is central to the airway EC migratory response and repair program (1,29). EGFR itself is sialylated (6), its sialylation state regulates receptor dimerization and activation (41), and its response to ligands can be influenced by gangliosides (7,8).…”

“…ϭ 100) after which they were incubated for 10 min with 100 ng/ml EGF or medium alone. The ECs were lysed, and the lysates were processed for phospho-EGFR (Tyr-1068), total EGFR, and ␤-tubulin immunoblotting as described (29). The phospho-EGFR blots were stripped and reprobed for total EGFR and ␤-tubulin, and the phospho-EGFR Tyr-1068 signal was normalized to ␤-tubulin in the same lane of the same gel.…”

Morphine Modulation of Thrombospondin Levels in Astrocytes and Its Implications for Neurite Outgrowth and Synapse Formation

“…To this end, we measured acute morphine-induced ERK phosphorylation in the presence of the function-blocking TSP1 Ab C6.7 (72). As seen in Fig.…”

Acute and Chronic Mu Opioids Differentially Regulate Thrombospondins 1 and 2 Isoforms in Astrocytes

“…In contrast, chronic (6 h) morphine may be a biased ligand for TSP1 degradation that results in PLC2 activation. 54 It would also be interesting to determine whether the functional selectivity arises from differential G protein or β-arrestin signaling. Since DAMGO but not morphine induces Oprm internalization, DAMGO may have a bias for β-arrestin signaling whereas morphine signals through G protein coupling.…”