Complementary DNA encoding the Na/Ca,K‐exchanger was isolated from bovine retina cDNA libraries. The complete full‐length cDNA is approximately 4 kb long and contains an open reading frame of 3597 bp. The deduced amino acid sequence corresponds to a protein of 1199 amino acids with a calculated molecular weight of approximately 130 kDa. Hydrophobicity analysis revealed the presence of two alternating sets of hydrophobic and hydrophilic domains. There also exists a hydrophobic region at the N‐terminus which may be part of a cleavable signal peptide. The protein shares limited sequence homology with the Na/Ca‐exchanger from cardiac sarcolemma. Northern blot analysis indicates that the approximately 6 kb transcript is highly specific for retinal tissue. Insect cells infected with recombinant baculovirus bearing the full‐length cDNA express a functional Na/Ca,K‐exchanger with an apparent relative molecular weight of approximately 210 kDa, as determined by Western blotting.

The lipid composition of two different insect cell lines from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) which are established cell lines for infection with recombinant baculovirus was analyzed by high-performance liquid chromatography and gas-liquid chromatography. The major phospholipids found were phosphatidylcholine and phosphatidylethanolamine, the major mono-unsaturated fatty acids were oleic acid and palmitoleic acid, the major saturated fatty acid was stearic acid. The cholesterol to phospholipid ratio was demonstrated to be lower than in mammalian cell lines. Infection with a recombinant baculovirus Autographa californica resulted in increased levels of phosphatidylcholine in the insect cells. The baculovirus/insect cell system has become a popular system for heterologous protein production. Functional changes of membrane proteins produced in these two cell lines might be correlated to a different lipid profile of their cellular membranes.z 1998 Federation of European Biochemical Societies.

We have expressed a c-myc epitope-tagged human oxytocin receptor in the baculovirus/Sf9 cell system. The receptor was identified by SDS-PAGE and subsequent immunoblot as a approximately 50 kDa protein which decreased to about 44 kDa upon treatment with tunicamycin. Binding studies showed that the human oxytocin receptor was expressed in a low-affinity state (Kd = 215 nM; Bmax = 1.66 pmol/mg). After addition of cholesterol in the form of a soluble cholesterol-methyl-beta-cyclodextrin complex to the membranes, we obtained part of the human oxytocin receptor in its high-affinity state for oxytocin (Kd = 0.96 nM and Bmax = 318 fmol/mg of protein). In subsequent studies, we added the cholesterol-methyl-beta-cyclodextrin complex to the Sf9 cell culture medium at various times post infection. Binding analysis showed that this results in a more than 3-fold further increase in functional receptor binding sites of high-affinity state (Bmax = 1.08 pmol/mg). The cholesterol effect was dose-dependent, with an EC50 of about 50 microM cholesterol. Due to these findings, we determined the cholesterol and phospholipid content in purified Sf9 plasma membranes. The untreated naturally cholesterol auxotroph insect cells grown in medium with 2% fetal calf serum had a molar cholesterol/phospholipid ratio of about 0.04, which is approximately 20-fold lower than normally found in plasma membranes of higher eukaryotic cells. The high-affinity binding of the oxytocin receptor increased in parallel with the cholesterol levels present in the corresponding plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTP belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTP. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTP ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTP ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTP at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.The phosphorylation state of numerous signaling proteins is controlled by opposing activities of protein-tyrosine kinases and protein-tyrosine phosphatases (PTP) 1 (1). The family of PTPs consists of soluble and receptor-like PTPs (RPTPs) (2). Whereas the intracellular region of RPTPs is relatively similar in all representatives containing either a single or two PTP domains, the extracellular region has a large diversity. PTP belongs to subclass IIB, called "MAM-containing PTP" (2). Besides the MAM domain (meprin/A5-protein/PTPmu domain; Ref.3), their extracellular region contains a single immunoglobulin (Ig)-like domain and four fibronectin (FN) III repeats (4). This structural architecture of ectodomain is similar to members of the cell-adhesion molecule superfamily.PTP is strongly expressed in the endothelial cell layer of the arteries and continuous capillaries as well as in cardiac muscle, bronchial and lung epithelia, retina, and several brain areas (4 -6). At the subcellular level, it is localized at sites of cell-cell contact (7). In this regard, it has been demonstrated that PTP restores E-cadherin-mediated cellular adhesion, when it is expressed in LNCaP human prostate carcinoma cells (8). Physiologically, PTP has been shown to be involved in promotion and regulation of neurite outgrowth (5, 9).Numerous experiments have clearly demonstrated that the extracellular region of PTP promotes cell-cell aggregation in a Ca 2ϩ -independent manner (10, 11). The homophilic binding has been also evidenced in the ectodomains of PTP (12) and PTP (13), strongly suggesting that these RPTPs may be involved in signal transduction through cell-to-cell contact in vivo. Evidence concerning the physiological role of PTP-mediated homophilic binding has been reported in a recent article (14) showing that homophilic interactions trigger rearrangements of the axonal growth cone. However, the molecular mechanism of this interaction remains larg...

In order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous D2S receptor, we have expressed various D2S receptor constructs in two Spodoptera frugiperda cell lines, a Trichoplusia ni and a Mammestra brassicae cell line. To improve expression yield, the environment of the polyhedrin gene translational initiation site was retained by fusing the first 12 codons of the polyhedrin gene to the 5'-end of the D2S receptor coding sequence. The pharmacological profile of the expressed D2S receptor was similar to that reported for neuronal D2 receptors. Sf9 and Tn cells were best suited for overexpression, yielding about 2 x 10(6) and 4 x 10(6) receptors/cell, respectively, corresponding to 6 pmol/mg of cell protein in Sf9 cells and 10 pmol/mg of cell protein in Tn cells. We have developed a D2 receptor-specific anti-peptide antibody to study glycosylation, palmitoylation, and localization of the heterologously produced receptor. Immunoprecipitation of digitonin/cholate-solubilized receptor from control and tunicamycin-treated Sf9, Tn, and Mb cells revealed an apparent molecular mass of 47-48 kDa for the glycosylated receptor and of 39-40 kDa for the unglycosylated receptor. Although pulse-chase studies showed that glycosylation occurred rapidly and efficiently, the glycosylated receptor only constituted a small fraction of the overall produced receptor protein, which was mainly located intracellularly. The glycosylation of the receptor was of the high-mannose-type in contrast to the complex-type glycosylation found in native tissue. The glycosylated D2S receptor was palmitoylated. Glycosylation, however, was not a prerequisite for palmitoylation which was insensitive to tunicamycin, brefeldin A, and monensin. NH2-terminal addition of the signal sequence of prepromelittin to the D2S receptor increased expression levels 2-3-fold and significantly enhanced membrane insertion and processing, resulting in increased targeting of the synthesized receptor to the plasma membrane.