Transient Association of the Endoplasmic Reticulum Protein, NogoB, with Integrin Complexes Provides a Novel Mechanism for Inducing Endothelial Cell Motility.
Authors: Bialkowska Katarzyna , Fox Joan E.B. 
Published: October 2019

Quiescent endothelial cells respond rapidly to pathological and physiological stimuli with integrin-mediated changes in actin filament content and organization and changes in cell motility. Little is known about mechanisms regulating the differential activation of the RhoGTPases that induce actin polymerization and determine their function and organization in integrin signaling complexes. Since the specificity of RhoGTPase recruitment is determined by adaptor proteins, we initiated studies to identify adaptor proteins that associate with integrin complexes in a regulated manner. Using bovine aortic endothelial (BAE) cells, we showed that spectrin, which contains an SH3 motif, is selectively recruited to transient integrin complexes that activate Rac and initiate membrane projections at new sites of attachment. In a two-hybrid screen, the spectrin SH3 domain interacted with a 9 proline-motif sequence in a protein of Mw ∼48 kD. The presence of multiple proline motifs suggested that this protein might be an adaptor, capable of interacting with spectrin and recruiting additional SH3-containing proteins to integrin complexes. Although it lacked 30 N-terminal residues, the sequence of the SH3-binding protein was otherwise identical to that of the endothelial cell protein, Nogo B. Nogo family members localize to the endoplasmic reticulum (ER), although some are in the plasma membrane, where their extracellular domain interacts with adjacent cells, inhibiting their growth. Despite 9 cytoplasmic proline motifs, the possibility that Nogo proteins regulate signaling in the cell in which they are expressed has not been investigated. In the present study, we determined whether Nogo B interacts with SH3-motifs in vivo and regulates signaling in integrin complexes. Using an SH3 domain array, we identified CrkII, Vav2 and intersectin as Nogo B-interacting proteins. In vivo, Nogo B recruited both spectrin and CrkII, as shown by their coimmunoprecipitation from BAEC with EGFP-48kD Nogo B. Both endogenous and EGFP-48 kD Nogo B localized to the population of spectrin-containing integrin complexes. Like spectrin, they were excluded from the focal adhesions that form in less motile cells, appearing to be present only in the ER in these cells. When expressed in BAEC, spectrin SH3 domain was recruited to the Nogo B containing integrin complexes, arresting Rac activation and membrane extension. To examine the role of NogoB in integrin-mediated motile events, NIH3T3 cells stably expressing EGFP-48k Da Nogo B or EGFP alone were plated on an integrin substrate; cells expressing 48 kDa Nogo B activated Rac, formed submembranous actin networks, extended projections, and spread more rapidly than control cells. The accelerated motile events did not occur in cells plated on poly-L-lysine, in cells treated with Nogo siRNA, in cells transfected with the 9 proline-motif sequence, or in cells transfected with the spectrin SH3 domain. They were initiated upstream of Rac activation, as shown by their absence in cells expressing dominant-negative Rac. These data suggest that Nogo proteins function as SH3-recruiting adaptors and describe a novel mechanism for accelerating integrin-mediated Rac activation and membrane projections through the transient association of the proline-rich sequence of Nogo B with the SH3 domain of integrin-associated spectrin, and recruitment of additional SH3-containing proteins involved in Rac activation to these integrin complexes.