Arrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule.Arrestins are multifunctional regulators of cell signaling (1, 2). Arrestins, which bind active phosphorylated G protein-coupled receptors (GPCRs), 2 which play a major role in receptor desensitization and internalization (3, 4). With the identification of numerous non-receptor binding partners, the classical paradigm of arrestin function has been expanded, implicating arrestins in mitogen-activated protein kinase (MAPK) activation, protein ubiquitination, chemotaxis, apoptosis, and other cellular functions (2, 5-11).The first indication that arrestins function as signaling adapters came from the studies of arrestin-dependent c-Src recruitment to the receptors, which results in the activation of extracellular signal-regulated kinases (ERK1/2) (10, 12, 13).Subsequently, arrestin2 and arrestin3 in complex with different receptors were reported to scaffold JNK3 (9), ERK1/2 (8, 14), and p38 (15, 16) activation cascades. Although arrestins play an important role in regulating different MAPK pathways, the mechanism of arrestin-dependent assembly of MAP kinases into a signaling complex remains largely unexplored. Existing models have limited predictive value. For example, the idea that JNK3 is activated solely by arrestin3 because this arrestin subtype has unique ability to bind JNK3 (9, 17) was not supported by further experimentation (18 -20). Similarly, the hypothesis that only receptor-bound arrestins interact with MAP kinases (8, 9) was not confirmed (17-20).Here we addressed several key mechanistic issues in arrestindependent MAPK signaling. First, we show that the scaffolding function is not limited to receptor-bound arrestin; free arrestin3 facilitates ASK1-mediated JNK3 activation, indicating that arrestins are not exclusively receptor-regulated adapters as thought previously. Second, we show that all four mammalian arrestins bind each component of the JNK3 cascade with comparable affinity, demonstrating that bindin...

Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus.

Arrestins are multifunctional signaling adaptors originally discovered as proteins that “arrest” G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β2-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the “constitutively inactive” arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.

-Inner medullary collecting ducts (IMCD) are terminally differentiated structures derived from the ureteric bud (UB). UB development is mediated by changes in the temporal and spatial expression of integrins and their respective ligands. We demonstrate both in vivo and in vitro that the UB expresses predominantly laminin receptors (␣ 3␤1-, ␣6␤1-, and ␣ 6␤4-integrins), whereas the IMCD expresses both collagen (␣1␤1-and ␣ 2␤1-integrins) and laminin receptors. Cells derived from the IMCD, but not the UB, undergo tubulogenesis in collagen-I (CI) gels in an ␣1␤1-and ␣2␤1-dependent manner. UB cells transfected with the ␣ 2-integrin subunit undergo tubulogenesis in CI, suggesting that collagen receptors are required for branching morphogenesis in CI. In contrast, both UB and IMCD cells undergo tubulogenesis in CI/ Matrigel gels. UB cells primarily utilize ␣ 3␤1-and ␣6-integrins, whereas IMCD cells mainly employ ␣ 1␤1 for this process. These results demonstrate a switch in integrin expression from primarily laminin receptors in the early UB to both collagen and laminin receptors in the mature IMCD, which has functional consequences for branching morphogenesis in three-dimensional cell culture models. This suggests that temporal and spatial changes in integrin expression could help organize the pattern of branching morphogenesis of the developing collecting system in vivo. kidney development; branching morphogenesis; three-dimensional culture; basement membranes THE MULTIBRANCHED INNER MEDULLARY collecting ducts (IMCD) of the kidney are highly ordered, terminally differentiated structures consisting of polarized epithelial cells derived embryologically from the ureteric bud (UB). The UB, which originates as an outgrowth from the Wolffian duct, also gives rise to the ureters and the epithelial lining of the trigone of the bladder. For such diverse urogenital segments to develop from the UB, cells must undergo well-defined differentiation processes (15).The collecting system of the kidney is formed by iterative branching morphogenesis of the UB. Although this process has been studied in whole animal, organ, and cell culture models, the molecular cues for its control are still poorly understood. Cell culture models, which predominantly utilize Madin-Darby canine kidney (MDCK) cells grown in three-dimensional (3D) collagen-I (3D-CI) gels, recapitulate branching morphogenesis in its simplest form. These models suggest that branching morphogenesis is a multistep process that requires sequential cell adhesion to extracellular matrix (ECM), cell spreading, cell proliferation, and cell migration to ultimately form multicellular tubelike structures (14). A balance between proliferation and apoptosis is critical for the final formation of the lumen of the tubule (5).Integrins, the predominant cell-surface receptors that mediate interactions between cells and ECM, play a crucial role in cellular functions such as cell adhesion, migration, proliferation, and apoptosis (7, 21). Both in vivo and in vitro data demonstrate the importance...