A novel widely expressed type of myosin (fifth unconventional myosin from rat: myr 5) from rat tissues, defining a ninth class of myosins, was identified. The predicted amino acid sequence of myr 5 exhibits several features not found previously in myosins. The myosin head domain contains a unique N‐terminal extension and an insertion of 120 amino acids at a postulated myosin‐actin contact site. Nevertheless, myr 5 is able to bind actin filaments in an ATP‐regulated manner. The head domain is followed by four putative light chain binding sites. The tail domain of myr 5 contains a region which coordinates two atoms of zinc followed by a region that stimulates GTP hydrolysis of members of the ras‐related rho subfamily of small G‐proteins. Myr 5 therefore provides the first direct link between rho GTPases which have been implicated in the regulation of actin organization and the actin cytoskeleton. It is also the first unconventional myosin for which a tail binding partner(s), namely members of the rho family, has been identified.
Retention of soluble proteins in the endoplasmic reticulum is dependent on their interaction with the KDEL (Lys-Asp-Glu-Leu) receptor in the Golgi apparatus and their subsequent retrieval back to the endoplasmic reticulum. We have studied the three-dimensional organization of the human KDEL receptor using sitedirected mutagenesis and sulfhydryl-specific labeling. We have identified four amino acid residues, Arg-5, Asp-50, Tyr-162, and Asn-165, which we suggest participate in the formation of the ligand binding pocket. Arg-5 and Asp-50 are shown to be located on the lumenal side of the membrane and are inaccessible from the cytoplasm. In addition, our results strongly support a topology of the KDEL receptor similar to the family of G-proteincoupled receptors with seven transmembrane domains. Furthermore, Asp-50 plays a crucial role in the binding of His/Lys-Asp-Glu-Leu ligands, but is not required for Asp-Asp-Glu-Leu binding, suggesting that this residue forms an ion pair with the positively charged amino acid residue positioned 4 residues from the carboxyl terminus of the ligand.The precise sorting of proteins along the secretory pathway is crucial for the maintenance of organelle integrity. The localization of chaperones and other soluble proteins to the ER 1 is achieved by their continuous retrieval from post-ER compartments by the KDEL receptor, a membrane protein localized in the Golgi apparatus. Binding of soluble ER proteins to the receptor is dependent on a conserved tetrapeptide sequence at their carboxyl terminus, which is usually KDEL in animal cells and HDEL in Saccharomyces cerevisiae (see Ref. 1 for review). The HDEL receptor was originally isolated from yeast; it is the product of the ERD2 gene and has subsequently been cloned from other organisms (2-6). Erd2 determines both capacity and specificity of the H/KDEL retrieval system (7), and a direct interaction between KDEL ligands and the human receptor has recently been demonstrated (8, 9). Hydrophobicity analyses and the "positive inside rule" (10) suggest seven primarily hydrophobic membrane-spanning domains with the amino terminus in the lumen and the carboxyl terminus in the cytoplasm (11,12). The topology of the KDEL receptor would thus be similar to the organization of G-protein-coupled receptors, although no amino acid sequence homology is observed. Based on studies using fusions to N-linked glycosylation sites, a different topology has been proposed with six transmembrane domains and the amino terminus located in the cytoplasm (13).Although the KDEL recycling system is well established, several significant questions have remained unanswered. For example, the sorting of KDEL proteins is believed to be controlled by the pH difference between the Golgi apparatus and the ER (8). However, it is unclear whether the pH difference between these two compartments, which is estimated to be approximately 0.5 pH units (8), is sufficient to ensure efficient binding of KDEL proteins in the Golgi apparatus and their release in the ER. In addition, ligand b...
Retention of soluble endoplasmic reticulum (ER) proteins is ensured by their continuous retrieval from subsequent compartments in the secretory pathway. Soluble ER proteins which escape to the Golgi apparatus bind to the KDEL receptor, a seven-transmembrane receptor, and are then returned to the endoplasmic reticulum. We have overexpressed the human KDEL receptor in insect cells using the baculovirus system. Infected cells accumulate large amounts of functional receptor as judged by a ligand binding assay. A hexahistidine-tagged version of the receptor could be purified in a single step to near homogeneity with high yield. After reconstitution of purified KDEL receptor into liposomes, a similar affinity and pH dependence for the binding of KDEL peptides was observed compared to the receptor in its natural environment, indicating that purified KDEL receptor is sufficient for specific and pH-sensitive binding of KDEL ligands. Determination of the receptor affinity in different lipid environments revealed that the receptor affinity is only slightly influenced by its lipid environment, suggesting that regulation of the receptor affinity by its surrounding lipids does not play a crucial role for the sorting of KDEL proteins.
Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and beta(2)-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1 microl without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.
Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and beta(2)-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1 microl without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.
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