Abstract. Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity-like the cationization strategy-as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics.

FSH receptor is selectively expressed on the surface of the blood vessels of a wide range of tumors. (Funded by INSERM.).

The complementary DNA for human thyroid-stimulating hormone (TSH) receptor encodes a single protein with a deduced molecular mass of 84.5 kDa. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (a subunit of = 53 kDa and p subunit of = 38 kDa) held together by disulfide bridges [Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. & Milgrom, E. (1992) Proc. Nutl Acad. Sci. USA 89, 3765-37691. A similar processing occurs in an L cell line permanently expressing the human TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95-kDa high-mannose precursor which is present only in trace amounts in the thyroid. Pulse-chase experiments show the successive appearance in the L cells of two precursors: initially the = 95-kDa high-mannose glycoprotein followed by a = 120-kDa species containing mature oligosaccharides. This latter precursor is then processed into the a and p subunits. In primary cultures of human thyrocytes precursors of similar size are detected.Spodopteru frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthesize a monomeric protein of about 90 kDa soluble only in denaturing conditions. Comparison with the product of in vitro transcription-translation experiments (= 80 m a ) , suggests that it may be incompletely or improperly glycosylated. The TSH receptor expressed in these cells is unable to bind the hormone.Immunoelectron microscopy studies show that in human thyrocytes most of the receptor is present on the cell surface; in L cells the receptor is detected on the cell surface, as well as in the endoplasmic reticulum and in the Golgi apparatus (this intracellular pool of receptor molecules probably corresponding to the high-mannose precursor) ; in insect cells nearly all the receptor molecules are trapped in the endoplasmic reticulum. These differences in receptor distribution are concordant with the differences observed for receptor processing.The thyroid-stimulating hormone (TSH) receptor has been the subject of extensive studies (reviews in [l, 21). Interest in this receptor stems not only from its key role in the control of thyroid function and growth (review in [3]), but also from its direct implication in autoimmune diseases. Autoantibodies against the TSH receptor display either a stimulatory effect and mimic the action of the hormone, provoking Graves' disease, or a blocking effect and lead to idiopathic myxoedema (reviews in [l, 2, 4, 51). However, due to its fragility and scarcity, attempts to purify the TSH receptor have been unsuccessful. Conflicting results have been reCorrespondence to E. Milgrom, HBpital de BicCtre, 3kme niveau, F-94275 Kremlin-Bicstre, FranceAbbreviations. TSH, thyroid stimulating hormone ; TSHR, thyroid stimulating hormone receptor; Sf, Spodopteru frugiperdu insect cells ; AcMNPV, Autogrupha Culifornicu multiple nuclear polyhedrosis virus ; D...

Scant information is available to date on the intracellular trafficking of the TSH receptor. In the present study we have used stably transfected L cells that express the TSH receptor, 225I-labeled TSH, and antireceptor antibodies as well as gold-conjugated antireceptor monoclonal antibodies and hormone. The latter allowed us to study, by electron microscopy, the cellular distribution and endocytosis of TSH receptor. The receptor was initially localized on the plasmalemma proper and in clathrin-coated pits but was excluded from smooth vesicles open to the cell surface. It was internalized through clathrin-coated vesicles. Constitutive endocytosis represented 10% of cell surface receptor molecules. Endocytosis was increased 3-fold by incubation with hormone. The majority of internalized receptor molecules (90%) was recycled to the cell surface, whereas the hormone was degraded in lysosomes. This recycling of receptor was inhibited by administration of monensin. Electron microscopic and confocal microscopic studies were repeated in primary cultures of human thyroid cells and showed a distribution, and endocytosis pathways, very similar to those observed in transfected L cells. A previous study has shown the LH receptor to be endocytosed in high proportion and to be degraded in lysosomes. Confocal microscopy and colocalization studies with transferrin receptor confirmed that the highly homologous LH and TSH receptors exhibit, when expressed in the same cells, very different cellular trafficking properties. The use of LH/TSH receptor chimeras showed that transmembrane-intracellular domains contain information orienting the protein toward recycling or degradative pathways. The extracellular domain seems to play a role in the extent of intemalization. These observations should now allow the identification of the molecular signals involved.