Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor ␣1 (GFR␣1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF 2 ⅐GFR␣1 2 complex structure at 2.35 Å resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF 2 ⅐GFR␣1 2 complex and the previously published artemin 2 ⅐GFR␣3 2 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR␣ interface between the two structures, in particular ones that buttress the key conserved Arg GFR␣ -Glu ligand -Arg GFR␣ interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR␣s, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand 2 -co-receptor 2 -RET 2 heterohexamer complexes. Consistent with this, we showed that GDNF 2 ⅐GFR␣1 2 and artemin 2 ⅐GFR␣3 2 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR␣1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg 190 -Lys 202 region in GFR␣1 domain 2. This may explain how GDNF⅐GFR␣1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET. GDNF,3 originally characterized as a growth factor promoting the survival of midbrain dopaminergic neurons (1), regulates the differentiation and development of many peripheral neurons (2) and is neuroprotective (3). GDNF is also a morphogenic factor in kidney and spermatogonia development (reviewed by Airaksinen and Saarma (2)). Some clinical trials have indicated that perfusing GDNF into the putamen may be therapeutically beneficial in Parkinson disease (4). These neuroprotective and therapeutic roles have generated wide interest in the study of the GDNF signaling system.There are three other GDNF family ligands (GFLs), neurturin (NRTN (5)), artemin (ARTN (6)), and persephin (PSPN (7)), and knock-out mice experiments have made it clear that the order of biological importance is GDNF Ͼ Ͼ NRTN Ͼ ARTN Ͼ PSPN (2). They all signal primarily through the receptor tyrosine kinase RET (8). The extracellular region of RET has four cadherin-like domains and a cysteine-rich domain. Mutations in RET can cause both gain-of-function and loss-of-function diseases. In the former category are hereditary medullary thyroid carcinoma and multiple endocrine neoplasias of types 2A and 2B (9, 10), whereas Hirschsprung disease is an example of the latter (10).GFLs are distant relatives of transforming growth factor ␤ (2). Each GFL has its own co-receptor ␣: GDNF requires GFR␣1; N...

State University, East Lansing, Michigan 48824-1 31 2 (V.K., N.V.R., S.G.)Previous work suggested that the aspartic proteinase from Hordeum vulgare (HvAP) would be a vacuolar protein in plant cells. Based on N-terminal sequencing we show that the in vitro-translated protein was translocated into the lumen of microsomal membranes, causing a concomitant removal of 25 amino acid residues from the protein. Vacuoles were purified from barley leaf protoplasts and were shown to contain all of the aspartic proteinase activity found in the protoplasts. This vacuolar localization of HvAP was confirmed with immunocytochemical electron microscopy using antibodies to HvAP in both barley leaf and root cells. In an attempt to discern a function for this protease, we investigated the ability of HvAP to process the C-terminal proregion of barley lectin (BL) in vitro. Prolectin (proBL), expressed in bacteria, was processed rapidly when HvAP was added. Using severa1 means, we were able to determine that 13 amino acid residues at the C terminus of proBL were cleaved off, whereas the N terminus stayed intact during this incubation. lmmunohistochemical electron microscopy showed that HvAP and BL are co-localized in the root cells of developing embryos and germinating seedlings. Thus, we propose that the vacuolar HvAP participates in processing the C terminus of BL.Proteolysis plays an important role in many biological processes, including protein degradation and tumover, protein processing, and pathogen attack. Proteinases have been classified as to the amino acid residues or cofactors involved in the proteinase activity, and the classification is usually based on the sensitivity of the activity to various specific inhibitors. The main classes include Ser proteinases, Cys proteinases, metalloproteinases, and Asp proteinases. There are examples of each of these types of proteinases in plants.Asp proteinases (EC 3.4.23) are a class of endopeptidases that are active at acidic pH, inhibited by pepstatin A, and show a conserved three-dimensional structure with two Asp residues in the active cleft (Tang and Wong, 1987; Davies, 1990). This group of proteinases is generally in the secretory pathway and is activated either outside the cell or inside the lysosome/vacuole (Tang and Wong, 1987; Davies, 1990). In

Two enzymatically active heterodimeric forms of an aspartic proteinase, a putative 32 kDa + 16 kDa precursor form and a putative 29 kDa + 11 kDa mature form, are present in resting barley grains (Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J. & Mikola, L., 1990, in the press). The cDNA corresponding to this enzyme has been cloned and sequenced. The full-length 1863-bp cDNA sequence codes for an open reading frame of 508 amino acids. The open reading frame consists of a 66-amino acid preprosequence and a 442-amino acid mature protein.Comparison of the N-terminal amino acid sequences of the enzyme subunits with the sequence of the cDNA clone indicates that the heterodimeric enzyme is translated as a proenzyme which is processed into two subunits. The localisation of the experimentally determined N-terminal amino acid sequences of all four subunits (32 kDa + 16 kDa and 29 kDa + 11 kDa) in the same transcript, as well as the detection of only one 2.0-kb mRNA on Northern blots from resting seeds, clearly indicates that the larger (32 kDa + 16 kDa) enzyme is an intermediate precursor form of the smaller (29 kDa + 11 kDa) enzyme. The processing pattern of the barley enzyme, which is the first sequenced plant aspartic proteinase, differs from that of all other known aspartic proteinases. The barley enzyme is highly similar to mammalian and yeast aspartic proteinases, especially to human and porcine cathepsin D. This similarity is clearly dispersed over two regions, separated by a dissimilar, barley-specific region of 104 amino acids.In animals and yeast, members of the family of aspartic proteinases, including cathepsin D, renin, chymosin and proteinase A, are very substrate-specific enzymes with well-defined physiological functions. This class of proteinases is involved in the activation of other enzymes, e.g. the lysosomal acid phosphatase and carboxypeptidase Y [l -31 as well as extracellular proteins like procollagen and the prohormonal angiotensinogen [4, 51. Aspartic proteinases are also involved in the digestion and inactivation of specific proteins like the protcinase-inhibitor cystatin and the IC chain of the milk Kcasein [6, 71. A common feature of aspartic proteinases is an active cleft where two aspartate residues play a central role in the catalytic mechanism [S, 91. The amino acid sequences around these aspartate residues are generally conserved. The Note. The novel nucleotide acid sequence data published here has been dcposited with the EMBL sequence data bank and i s available under accession number X56136.Dediwtion. To the memory of Juhani Mikola who initiated this work as a continuation of his research on storage proteins and their mobilisation during germination. activity of most aspartic proteinases is inhibited by the specific inhibitor pepstatin. Different aspartic proteinases differ in their cellular localisation and processing pattern, as well as in their substrate specificity.In plants, aspartic proteinase activity has been localised in seeds from a broad variety of plant sp...

RET (REarranged during Transfection) is a transmembrane receptor tyrosine kinase that is activated by a complex consisting of a soluble glial cell line-derived neurotrophic factor (GDNF) family ligand (GFL) and a glycosyl phosphatidylinositol-anchored co-receptor, GDNF family receptors alpha (GFRalpha). RET signalling is crucial for the development of the enteric nervous system. RET also regulates the development of sympathetic, parasympathetic, motor, and sensory neurons, and is necessary for the postnatal maintenance of dopaminergic neurons. The effect of GFLs on sensory, motor, and dopaminergic neurons has raised clinical interest towards these ligands. Outside the nervous system, RET is crucial for development of the kidney and plays a key role in spermatogenesis. Inactivating mutations in RET cause the Hirschsprung's disease characterized by megacolon aganglionosis. In contrast, activating mutations give rise to different types of cancer, multiple endocrine neoplasia type 2A and type 2B, familial medullary thyroid carcinoma, and papillary thyroid carcinoma. The multiple disease phenotypes correlate with differences in the molecular and cell biological functions of different oncogenic RET proteins. In this review we summarize how the different domains of the RET protein contribute to its normal function and how mutations in these domains affect the function of the receptor.