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Caveolae are lipid raft microdomains that regulate endocytosis and signal transduction. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates caveolin 1, supporting a role for these subcellular regions in the compartmentalization of IGF-I signaling. Src homology 2/alpha-collagen related protein (Shc) is the main mediator of IGF-I mitogenic action, coupling IGF-IR phosphorylation to Ras-MAPK activation. Here we show that IGF-I induces Shc tyrosine phosphorylation in the caveolae with a time course significantly different from that observed in the nonraft cellular fractions. In the same time, IGF-I recruits growth factor receptor bound protein 2 (Grb2) to caveolae and activates p42/p44 MAPKs in these microdomains. Src family kinases regulate IGF-I action through an Shc-dependent mechanism. In R-IGF-IRWT cells, IGF-I causes Fyn enrichment in the caveolae with a time course consistent with Shc phosphorylation and Grb2 recruitment in these regions. Finally, we have observed that after IGF-I stimulation, IGF-IR and Fyn colocalize in lipid raft caveolin 1-enriched microdomains. As insulin and IGF-I share common substrates, the effect of insulin on these cellular processes was measured. Here we show that insulin also induces Shc phosphorylation and Grb2 recruitment to caveolae, but with a significantly different time course compared with IGF-I. Our results suggest that 1) IGF-I causes the colocalization of signaling proteins in caveolae through a phosphorylation-regulated mechanism; and 2) the time course of phosphorylation and recruitment of substrates in caveolae by insulin receptor and IGF-IR could determine the specific actions of these receptors.

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.

The extracellular regions of insulin and IGF-I receptors (IR and IGF-IR) contain fibronectin type III repeats with cysteine residues potentially involved in S==S bond. In this report we show that Cys 786 in the IR and the corresponding Cys 776 in the IGF-IR regulate proreceptor dimerization with high specificity. Both C786S insulin and C776S IGF-I proreceptors reach the monomeric 210-kDa step, but do not proceed further. Mature IR(C786S) and IGF-IR(C776S) expression on plasmamembrane is abolished. No retention of C786S IR precursor was detected in the endoplasmic reticulum, which is degraded by a nonlysosomal mechanism. The rearrangement of the remaining cysteines in the insulin receptor beta subunit ectodomain does not rescue dimerization of C786S insulin proreceptor. As observed in other transmembrane receptors, iuxtamembrane cysteines, specifically Cys 786 in the IR and Cys 776 in the IGF-IR, are critical for correct processing of proreceptors., (Copyright 2001 Academic Press.)

Previous work has shown that red wines, grape juices, and other grape products cause endothelium-dependent relaxation (EDR) of blood vessels in vitro by increasing nitric oxide production. In this paper we describe the isolation and characterization of some of the compounds responsible for EDR activity. Concord grape seeds were extracted with methanol and the compounds were separated by Toyopearl TSK HW-40S chromatography. Resulting fractions (primarily phenolic acids, catechins, and proanthocyanidins) were further separated semipreparatively by reversed-phase HPLC, and peaks were collected and bioassayed for EDR activity using the rat aorta preparation. EDR-active compounds were subsequently characterized by HPLC retention times and electrospray-ion-trap mass spectrometry. The compounds exhibiting the most EDR activity were proanthocyanidin trimers, tetramers, pentamers, and polymers and their gallates, as well as a dimer gallate (EC50 values in the range of 0.6-2.5 microg catechin equivalents/mL). These compounds should be useful for in vitro and in vivo studies, particularly as they relate to improvement of cardiovascular function.

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.

We investigated the effects of a ser for cys 860 mutation, located in the extracellular portion of insulin receptor beta subunit, on several receptor functions. CHO cells, stably transfected with the mutated cDNA, were used for this study. In the present paper, we show that the ser 860 mutation does not affect the 125I-insulin binding, but severely impairs the insulin-insulin receptor complex internalization. The kinetic analysis of internalization indicates that this process is inhibited at steps preceding the coated pit endocytosis. The beta subunit autophosphorylation of the mutated receptor is higher both in the basal and insulin stimulated states, compared with autophosphorylation measured in wild type insulin receptors. The ser 860 mutation impairs also the insulin receptor down regulation, thus suggesting an effect on the intracellular sorting of insulin-insulin receptor complex. On the basis of these results we suggest that the cys 860 plays an important role in insulin receptor lateral moving on cell surface, after insulin binding, and on the intracellular sorting to degradation pathways.