BPAG1 isoform-b: complex distribution pattern in striated and heart muscle and association with plectin and alpha-actinin.

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

Dimerization of Kit-ligand and efficient cell-surface presentation requires a conserved Ser-Gly-Gly-Tyr motif in its transmembrane domain.

Kit-ligand (Kitl), also known as stem cell factor, is a membrane-anchored, noncovalently bound dimer signaling via the c-kit receptor tyrosine kinase, required for migration, survival, and proliferation of hematopoietic stem and germ cells, melanocytes, and mastocytes. Despite its fundamental role in morphogenesis and stem cell biology, the mechanisms that regulate Kitl dimerization are not well understood. By employing cell-permeable cross-linker and quantitative bimolecular fluorescence complementation of wild-type and truncated forms of Kitl, we determined that Kitl dimerization is initiated in the endoplasmic reticulum and mediated to similar levels by the transmembrane and the extracellular growth factor domain. Further biochemical and mutational analysis revealed a conserved Ser-Gly-Gly-Tyr-containing motif that is required for transmembrane domain dimerization and efficient cell-surface expression of Kitl. A novel intracellular capture assay with the Kitl transmembrane domain as bait revealed specific interactions with Kitl, but not with unrelated transmembrane proteins. During evolution, the transmembrane dimerization motif appeared in Kitl at the transition from teleosts to tetrapods, which correlates with the emergence of Kitl as a supporter of stem cell populations. Thus, transmembrane-mediated association of membrane-anchored growth factors consists of a novel mechanism to improve paracrine signaling and morphogenesis.

The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells.

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

A specific endoplasmic reticulum export signal drives transport of stem cell factor (Kitl) to the cell surface.

Stem cell factor, also known as Kit ligand (Kitl), belongs to the family of dimeric transmembrane growth factors. Efficient cell surface presentation of Kitl is essential for the migration, proliferation, and survival of melanocytes, germ cells, hemopoietic stem cells, and mastocytes. Here we demonstrate that intracellular transport of Kitl to the cell surface is driven by a motif in the cytoplasmic tail that acts independently of the previously described basolateral sorting signal. Transport of Kitl to the cell surface is controlled at the level of the endoplasmic reticulum (ER) and requires a C-terminal valine residue positioned at a distance of 19-36 amino acids from the border between the transmembrane and cytoplasmic domains. Deletion or substitution of the valine with other hydrophobic amino acids results in ER accumulation and reduced cell surface transport of Kitl at physiological expression levels. When these mutant proteins are overexpressed in the ER, they are transported by bulk flow to the cell surface albeit at lower efficiency. A fusion construct between Kitl and the green fluorescent protein-labeled extracellular domain of a temperature-sensitive mutant of vesicular stomatitis virus G protein revealed the valine-dependent recruitment into coat protein complex II-coated ER exit sites and vesicular ER to Golgi transport in living cells. Thus the C-terminal valine defines a specific ER export signal in Kitl. It is responsible for the capture of Kitl at coat protein complex II-coated ER exit sites, leading to subsequent cell surface transport under physiological conditions.

Phosphoinositide 3-kinase C2alpha is activated upon smooth muscle cell migration and regulated by alpha(v)beta(3) integrin engagement.

The involvement of phosphoinositide 3-kinase C2alpha in vascular smooth muscle cell migration was investigated. Products of phosphoinositide 3-kinase, phosphatidylinositol-3-phosphate, and phosphatidylinositol-3,4-bis-phosphate were increased upon smooth muscle cell migration but their synthesis was affected only partially by phosphoinositide 3-kinase inhibitors, wortmannin and LY-294002. Using specific antibody, we showed that the wortmannin/LY-294002 poorly sensitive phosphoinositide 3-kinase C2alpha is expressed in smooth muscle cells. Measurement of phosphoinositide 3-kinase C2alpha activity in vitro, after immunoprecipitation, clearly demonstrated its activation upon smooth muscle cell migration. Moreover, for the first time, phosphoinositide 3-kinase C2alpha was found to be differentially regulated by alpha(v)beta(3) and alpha(v)beta(5) integrin engagement. Finally, we have identified two new potential phosphoinositide 3-kinase C2alpha-binding proteins, p70 and p110, which both may be tyrosine phosphorylated. Thus, phosphoinositide 3-kinase C2alpha might represent a new regulatory pathway of cell migration downstream of integrin engagement.