Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R.caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.     

Affinity labeling by a photoreactive GTP analogue of the alpha-subunit of eukaryotic initiation factor eIF-2 in different initiation complexes

The interaction of GTP with initiation factor eIF-2 in different complexes was studied by affinity labeling using a derivative of [3H]GTP carrying a photoreactive group in the alpha-phosphate moiety. In the binary complex [eIF-2.GTP analogue], in the ternary complex [eIF-2.GTP analogue.Met-tRNAf] as well as in the eIF-2. eIF-2B complex the alpha-subunit of eIF-2 was found to be specifically labeled. GTP is concluded to interact during polypeptide chain initiation with the alpha-subunit of eIF-2 at least by its alpha-phosphate group.     

VIP21, a 21-kD membrane protein is an integral component of trans-Golgi- network-derived transport vesicles

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.     

Amino acid sequence analysis of the beta- and gamma-subunits of eukaryotic initiation factor eIF-2. Identification of regions interacting with GTP

By affinity labelling using two different GTP photoaffinity analogues we previously demonstrated that both the beta- and gamma-subunits of eukaryotic initiation factor eIF-2 are involved in GTP binding (Bommer, U.-A. and Kurzchalia, T.V. (1989) FEBS Lett. 244, 323-327). We have now applied the same method in combination with CNBr cleavage and microsequence analysis in order investigate which part of the polypeptide chain of eIF-2 beta is in close contact to the bound GTP. From the three main CNBr fragments of eIF-2 beta, the C-terminal one was found to be labelled by the applied GTP photoaffinity analogue, Guo(2',3'-TDBH)ppp. Because the cDNA sequence of the gamma-subunit of eIF-2 has not yet been published and because cDNA sequence analysis of eIF-2 beta revealed only two out of three consensus sequence elements of a GTP-binding domain, we also sequenced the CNBr fragments of eIF-2 gamma. In this way, sequences containing about 50 amino acid residues were obtained. Taken together with the recently published N-terminal sequences of tryptic peptides of eIF-2 gamma from pig liver (Suzuki et al. 1990, J. Biochem. 108, 635-641), about 30% of the total sequence is now known. One of the CNBr fragments from rabbit eIF-2 gamma contains a sequence (AXXAXXGK) which in several respects resembles that of the consensus sequence element absent from the beta-subunit.     

Long‐Chain O‐Ascarosyl‐alkanediols Are Constitutive Components of Caenorhabditis elegans but Do Not Induce Dauer Larva Formation

Two long‐chain ascarosides, O‐ascarosylnonacosane‐2,28‐diol ( 1 ) and O‐ascarosyluntriacontane‐2,30‐diol ( 2 ), were isolated from Caenorhabditis elegans and detected in all developmental stages of the worm. The long‐chain ascarosides were shown to be minor lipid components, and it was also shown that they do not induce dauer larva formation.     

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediaesp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.     

Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro

Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.     

Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

INTRODUCTIONAsearlyasin1948wehavefr8CtionatedisolatednucleifromnormalandtumorcellsbyextractionwithiMNaCIanddilutealkali[1].Thenuclearresiduewasthenstudiedmorethoroughly[2,3].Lateron,sillillarproteinousnuclearresidueswereisolatedbyotherworkers[46]andasstud…