Many cell surface proteins are attached to membranes via covalent glycosylphosphatidylinositol (GPI) anchors that are posttranslationally linked to the carboxy-terminus of the protein. Removal of the GPI lipid moieties by enzymes such as GPI-specific phospholipases or by chemical treatments generates a soluble form of the protein that no longer associates with lipid bilayers. We have found that the removal of lipid moieties from the anchor can also have a second, unexpected effect on the antigenicity of a variety of GPI-anchored surface molecules, suggesting that they undergo major conformational changes. Several antibodies raised against GPI-anchored proteins from protozoa and mammalian cells were no longer capable of binding the corresponding antigens once the lipid moieties had been removed. Conversely, antibodies raised against soluble (delipidated) forms reacted poorly with intact GPI-anchored proteins, but showed enhanced binding after treatment with phospholipases. In the light of these findings, we have reevaluated a number of publications on GPI-anchored proteins. Many of the results are best explained by lipid-dependent changes in antigenicity, indicating this might be a widespread phenomenon. Since many pathogen surface proteins are GPI-anchored, researchers should be aware that the presence or absence of the GPI lipid moieties may have a major impact on the host immune response to infection or vaccination.

To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.

GPEET procyclin is a major glycosylphosphatidylinositol-anchored protein of procyclic (insect stage) trypanosomes in culture and is heavily phosphorylated in the GPEET pentapeptide repeat. The phosphorylation reaction is a late event and occurs during maturation and transport of GPEET or on the parasite surface by an ecto-protein kinase. Initial biochemical characterization of the GPEET kinase activity now shows that it depends on bivalent cations for maximal activity, is stimulated by sulfhydryl group reagents, and is specific for ATP as phosphoryl donor. No kinase activity is detected in bloodstream form trypanosomes in culture, whereas strong phosphorylation is observed in early procyclic forms. In addition, the GPEET kinase activity is absent from procyclic trypanosomes that have repressed GPEET synthesis but can be induced in these same stocks by conditions, which also induce GPEET expression. However, the presence of an active kinase does not depend on the presence of (functional) GPEET because it can be detected in parasites expressing a non-phosphorylatable GPEET mutant protein and in procyclin null mutant trypanosomes. Interestingly, the presence of the glycosylphosphatidylinositol lipid moiety seems necessary for GPEET to become phosphorylated. Together, the results demonstrate that GPEET and its kinase are expressed during the same life cycle stages and that factors that induce the expression of GPEET in vitro also induce the expression of the GPEET kinase.

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The insulin receptor is composed of two u and two I subunits disulfide linked to form an u2f32 native structure. The ligand-binding ce subunit is extracellular, and the 3 subunit spans the membrane only once, thanks to a 23-amino acid hydrophobic domain, and contains a protein tyrosine-kinase region [1]. Although earlier studies had suggested that the transmembrane (TM) domain does not play a major role in the signal transduction process in the insulin receptor, evidence to the contrary has recently accumulated. Some modifications in this TM domain have been found to alter receptor internalization, negative cooperativity and insulin signaling. Strikingly, mutations resembling a wellcharacterized, activating mutation in the TM of the protooncogene tyrosine-kinase c-neu/erb-2, as well as the substitution of the insulin receptor TM domain by that of cneu/erb-2, lead to the complete or partial activation of the insulin receptor [1, 2]. Although the precise mechanism by which these modifications confer increased receptor activation is not known, a model implying increased dimerization and oligomerization by direct TM-TM interactions between uJ3 dirners has been proposed. Dimerization and oligomerization are though to be the primary events leading to activation of the intracellular tyrosine kinase in the case of the growth-factor receptors family.In order to study further the role of TM domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain by that of glycophorin A, a membrane protein unrelated to tyrosine-kinase receptors. Glycophorin A spontaneously forms detergentresistant dimers through TM-TM interactions. Residues in the TM domain of glycophorin A responsible for these interactions have been characterized by mutagenesis, and a specific dimerization driving amino acid pattern has been defined in this domain [3]. We have thus characterized the structural and functional properties of chimeric insulin receptors containing the TM domain of glycophorin A.An insulin receptor cDNA sequence was constructed with the insulin receptor TM domain replaced with the TM domain of glycophorin A (figure 1), and stably transfected in Chinese Hamster Ovary cells (CHO). Clona! cell lines expressing either wild-type insulin receptors (IR) or chimeric ones (IRGpA) were characterized for their '251-insulin binding properties, and compared to the parental wild type cell line (WT). Levels of expression of the chimeric receptors varied between different IRGpA-CHO clones (7 l0-3 10 sites/cell), as compared to 106 for the control IR-CHO cells, and 50000 for the wild-type cells. The apparent binding affinity constant was similar for all cell types. Insulin receptors were purified by WGA-agarose chromatography from IR-and IRGpA-CHO cells, and assayed for their insulin-stimulated autophosphorylation. Both types of receptors displayed similar insulin dose-response curves.The structure of these receptors was studied by SDS-PAGE electrophoresis after autophosphorylation, in the presence of inc...