The mammalian caveolin gene family consists of caveolins-1, -2, and -3. The expression of caveolin-3 is musclespecific. In contrast, caveolins-1 and -2 are co-expressed, and they form a hetero-oligomeric complex in many cell types, with particularly high levels in adipocytes, endothelial cells, and fibroblasts. These caveolin hetero-oligomers are thought to represent the functional assembly units that drive caveolae formation in vivo. Here, we investigate the mechanism by which caveolins-1 and -2 form hetero-oligomers. We reconstituted this reciprocal interaction in vivo and in vitro using a variety of complementary approaches, including the generation of glutathione S-transferase fusion proteins and synthetic peptides. Taken together, our results indicate that the membranespanning domains of both caveolins-1 and -2 play a critical role in mediating their ability to interact with each other. This is the first demonstration that these unusual membrane-spanning regions found in the caveolin family play a specific role in protein-protein interactions.

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S⅐mRNA⅐ eIF3⅐Met-tRNA f ⅐eIF2⅐GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32 P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude initiation factor preparations. We observed that eIF5 specifically interacted with the ␤ subunit of initiation factor eIF2. No other initiation factors including the ␥ subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the ␤ subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2␤ deletion mutants expressed in Escherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2␤. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNA f ⅐eIF2⅐GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the ␤ subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation.Initiation of translation in eukaryotic cells occurs by a sequence of partial reactions requiring the participation of a large number of specific proteins called eukaryotic (translation) initiation factors (eIFs).1 An obligatory intermediate step in this overall initiation reaction is the binding of the initiator methionyl-tRNA (Met-tRNA f ) as Met-tRNA f ⅐eIF2⅐GTP ternary complex to a 40 S ribosomal subunit, followed by positioning of the 40 S preinitiation complex (40 S⅐Met-tRNA f ⅐eIF2⅐GTP) at the initiation AUG codon of the mRNA to form the 40 S initiation complex (40 S⅐mRNA⅐Met-tRNA f ⅐eIF2⅐GTP). A 60 S subunit then joins the 40 S initiation complex to form the 80 S initiation complex (80 S⅐mRNA⅐Met-tRNA f ) that is active in peptidyl transfer (for a review, see Refs. 1-3). The subunit joining reaction specifically requires the participation of eIF5, an initiation factor that we have purified and characterized from mammalian cells (4 -6) and the yeast Saccharomyces cerevisiae (7). Detailed characterization of the eIF5-catalyzed reaction has shown that eIF5 first interacts with the 40 S initiation complex in the absence of 60 S ribosomal subunits to promote the hydrolysis of ribosome-bound GTP (8). Hydrolysis of GTP causes the release of eIF2 and guanine nucleotide (as an eIF2⅐GDP complex) from the 40 S subunit, an event that is essential for the subsequent joining of the 60 S ribosomal subunit to the 40 S complex (40 S⅐mRNA⅐Met-tRNA f ) to form the 80 S initiation complex that is active in subsequent peptidyl transfer reaction (8 -11).The mammalian cDNA (rat and human) and the S. cerevisiae gene encoding eIF5 of calculated M r ϭ 48,926 and 45,346, respectively, have been cloned and expressed in Escherichia coli (12)(13)(14)(15)). An interesting feature of the derived amino acid sequences of mammal...

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Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50^100 nm. Although recombinant expression of caveolin-1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin-caveolin interactions regulate the size of caveolae, as caveolin-1 undergoes two stages of self-oligomerization. To test this hypothesis directly, we have created two caveolin-1 deletion mutants that lack regions of caveolin-1 that are involved in directing the self-assembly of caveolin-1 oligomers. More specifically, Cav-1 v v61^100 lacks a region of the N-terminal domain that directs the formation of high molecular mass caveolin-1 homo-oligomers, while Cav-1 v vC lacks a complete C-terminal domain that is required to allow caveolin homo-oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav-1 v v61^100 and Cav-1 v vC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500^1000 nm. In addition, caveolin-induced vesicle formation appears to be isoformspecific. Recombinant expression of caveolin-2 under the same conditions failed to drive the formation of vesicles, while caveolin-3 expression yielded caveolae-sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin-1 expression, but continue to express caveolin-2, no morphologically distinguishable caveolae are observed. In addition, as caveolin-2 alone exists mainly as a monomer or homo-dimer, while caveolins 1 and 3 exist as high molecular mass homo-oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae-sized vesicles. In direct support of this notion, regulated induction of caveolin-1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin-2 to caveolae membranes. The ability of caveolin-1 to recruit caveolin-2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co-expressed and interact with each other to form high molecular mass hetero-oligomers containing both caveolins 1 and 2.z 1998 Federation of European Biochemical Societies.

Background:Definite etiology of amyotrophic lateral sclerosis (ALS) is still a matter of debate.Aims:The study was designed to evaluate the role of environmental, occupational, and familial risk factors in development of ALS.Materials and Methods:This was a case control study of 110 cases of definite ALS with 240 age and sex matched controls. Investigations were done on the following aspects- family history, occupation, living place, source of drinking water, exposure to industrial, chemical, agricultural toxins and heavy metals, physical and electrical injury, working under magnetic field for more than 10 years in both the groups. Clinical examinations, electrophysiological, and neuroimaging studies were done in every patient. Chi square test, logistic regression analysis, and calculation of odds ratio were used to analyze the data.Results:Rural livings (odds ratio = 1.99), smoking (odds ratio = 1.88), insecticides, and pesticides exposures (odds ratio = 1.61), electrical injury (odds ratio = 6.2) were detected as the associated factors in development amyotrophic lateral sclerosis.Conclusions:The study expressed the need of extensive research globally in molecular and genetic levels to detect the associated factors in etiopathogenesis of ALS for better understanding the etiology and for remedial aspects.

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with ␤-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and coimmunoprecipitates with a fusion protein containing the cytoplasmic tail of ␤-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of ␤-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within ␤-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with ␤-dystroglycan. In this regard, interaction of caveolin-3 with ␤-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy.

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