Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of ␤-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with ␤-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with ␤-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the ␤-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of ␤-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of ␤-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

Agonist-induced desensitization and resensitization of G-protein-linked receptors involve the interaction of receptors with protein kinases, phosphatases, ␤-arrestin, and clathrin organized by at least one scaffold protein. The dynamic composition of the signaling complexes and the role of the scaffold protein AKAP250 (gravin) in agonist-induced attenuation and recovery of ␤-adrenergic receptors were explored by co-immunoprecipitation of target elements, antisense suppression, and confocal microscopy. Gravin associated with unstimulated receptor, and the association was increased significantly after agonist stimulation for up to 60 min. Agonist stimulation also induced a robust association of the receptor-gravin complex with protein kinases A and C, G-protein-linked receptor kinase-2, ␤-arrestin, and clathrin. Confocal microscopy of the green fluorescence protein-tagged ␤ 2 -adrenergic receptor showed that the receptor underwent sequestration after agonist stimulation. Suppression of gravin expression via antisense oligodeoxynucleotides disrupted agonist-induced association of the receptor with G-protein-linked receptor kinase-2, ␤-arrestin, and clathrin as well as receptor recovery from desensitization. Gravin deficiency also inhibited agonist-induced sequestration. These data reveal that gravin-mediated formation of signaling complexes with protein kinases/phosphatases, ␤-arrestin, and clathrin is essential in agonist-induced internalization and resensitization of G-protein-linked receptors. G-protein-linked receptors (GPLRs)1 display attenuation of the receptor-mediated signal or "desensitization" and then "resensitization" after removal of agonist (1, 2). Protein phosphorylation is a critical element of agonist-induced desensitization involving at least three prominent kinase activities: cyclic AMPdependent protein kinase (protein kinase A), calcium-and phospholipid-sensitive protein kinase (protein kinase C), and members of the G-protein-linked receptor kinase family (GRK) such as the ␤-adrenergic receptor kinases (2). ␤ 2 -Adrenergic receptors are substrates for protein kinases A and C and Gprotein-linked receptor kinases as well as growth factor receptors with intrinsic tyrosine kinase activity (3, 4). Recent studies revealed that some G-protein-linked receptors such as the ␤ 2 -adrenergic receptor, m1 muscarinic cholinergic receptor, luteinizing hormone/human chorionic gonadotropin receptor, and gastrin-releasing peptide receptor undergo sequestration via clathrin-dependent endocytosis after agonist stimulation (5-7). ␤-Arrestin has been found to play an important role in targeting the receptors to clathrin-coated pits (8 -11). Following phosphorylation of the agonist-occupied receptors by GRKs, ␤-arrestin binds to the receptors, thereby terminating signaling transduction via endocytosis (10).Agonist-induced desensitization and sequestration of GPLRs are a complex process involving phosphorylation of the receptor by various protein kinases, followed by the interaction of the receptor with other proteins ...

Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg–Gly–Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10–100 nm. FN-null cells cultured on recombinant CCBD (FNIII8–11) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII8–11 contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII8–11 and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration.

Burns are dynamic injuries, characterized by progressive death of surrounding tissue over time. Though central to an understanding of burn injury progression, the spatiotemporal degrees and rates of cellular necrosis and apoptosis in the zone of ischemia surrounding burns are not well characterized. Using a validated porcine hot comb model, we probed periburn tissue at 1, 4, and 24 hours after injury for high mobility group box 1 (HMGB1) as a marker of necrosis and activated cleaved caspase 3 (CC3a) as a marker of apoptosis, followed by spatiotemporal morphometric analysis. We found that necrosis was the most prominent mechanism of cell death in burn injury progression, with significant progression between 1 and 4 hours post-burn. Apoptosis appeared not to play a role in early burn injury progression, but was observed in cells at the interface of necrotic and viable tissue at 24 hours post-burn. Our findings imply that intervention within the first 4 hours following injury is likely necessary to limit burn injury progression. Additionally, based on HMGB1 staining patterns, we define distinct early, intermediate and late pathological signs of cell necrosis that may facilitate delineation of causal mechanistic relationships of burn injury progression in vivo.