The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.
SummaryEph receptors are the largest subfamily of receptor tyrosine kinases regulating cell shape, movements and attachment. The interactions of the Ephs with their ephrin ligands are restricted to the sites of cell-cell contact since both molecules are membrane attached. This review summarizes recent advances in our understanding of the molecular mechanisms underlining the diverse functions of the molecules during development and in the adult organism. The unique properties of this signaling system that are of highest interest and have been the focus of intense investigations are as follows: (i) the signal is often simultaneously transduced in both ligand-and receptor-expressing cells, (ii) signaling via the same molecules can generate opposing cellular reactions depending on the context, and (iii) the Ephs and the ephrins are divided in two subclasses with promiscuous intra-subclass interactions, but rarely observed inter-subclass interactions.
SUMMARY Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer’s precursor protein, APP. We present here the X-ray crystal structure of the ADAM10 ectodomain, which together with biochemical and cellular studies reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture, in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.
Here we investigated how capping and methylation of HIV pre-mRNAs are coupled to Pol II elongation. Stable binding of the capping enzyme (Mce1) and cap methyltransferase (Hcm1) to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Both Mce1 and Hcm1 travel with Pol II during elongation. The capping and methylation reactions cannot occur until the nascent pre-mRNA has attained a chain length of 19-22 nucleotides. HIV pre-mRNAs are capped quantitatively when elongation complexes are halted at promoter-proximal positions, but capping is much less efficient during unimpeded Pol II elongation. Cotranscriptional capping of HIV mRNA is strongly stimulated by Tat, and this stimulation requires the C-terminal segment of Tat that mediates its direct binding to Mce1. Our findings implicate capping in an elongation checkpoint critical to HIV gene expression.
Human and fission yeast cDNAs encoding mRNA (guanine-N7) methyltransferase were identified based on similarity of the human (Hcm1p; 476 amino acids) and Schizosaccharomyces pombe (Pcm1p; 389 amino acids) polypeptides to the cap methyltransferase of Saccharomyces cerevisiae (Abd1p). Expression of PCM1 or HCM1 in S. cerevisiae complemented the lethal phenotype resulting from deletion of the ABD1 gene, as did expression of the NH 2 -terminal deletion mutants PCM1(94 -389) and HCM1(121-476). The CCM1 gene encoding Candida albicans cap methyltransferase (Ccm1p; 474 amino acids) was isolated from a C. albicans genomic library by selection for complementation of the conditional growth phenotype of S. cerevisiae abd1-ts mutants. Human cap methyltransferase was expressed in bacteria, purified, and characterized. Recombinant The m7GpppN cap of eukaryotic mRNA is formed by a series of three enzymatic reactions in which the 5Ј-triphosphate end of nascent pre-mRNA is hydrolyzed to a 5Ј-diphosphate by RNA triphosphatase, then capped with GMP by GTP:RNA guanylyltransferase, and methylated by RNA (guanine-N7) methyltransferase (1). RNA capping is essential for cell growth. Mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the yeast capping apparatus which abrogate catalytic activity are lethal in vivo (2-12).The physical and functional organizations of the capping apparatus differ in significant respects in fungi, metazoans, protozoa, and viruses. For example, fungi and mammals use distinct strategies to assemble a bifunctional enzyme with triphosphatase and guanylyltransferase activities. In yeast, separate triphosphatase (Cet1p) and guanylyltransferase (Ceg1p) polypeptides interact to form a heteromeric complex (10, 11, 13), whereas in mammals, autonomous triphosphatase and guanylyltransferase domains are linked in cis within a single polypeptide (Mce1p) (14, 15). The triphosphatase and guanylyltransferase components of the vaccinia virus and baculovirus capping enzymes also reside within single polypeptides (16 -19). The active sites of the guanylyltransferases are conserved among fungi, mammals, protozoa, and DNA viruses (6,18,20,21), but the triphosphatase components diverge with respect to structure and mechanism. There are at least two distinct classes of RNA triphosphatases: (i) the divalent cationdependent RNA triphosphatase/NTPase family (exemplified by yeast Cet1p, baculovirus LEF-4, and vaccinia D1) (11, 12, 16 -19, 21) and (ii) the divalent cation-independent RNA triphosphatases, e.g. the metazoan cellular enzymes and the baculovirus enzyme BVP (14,15,(22)(23)(24)(25)(26).The enzyme RNA (guanine-N7) methyltransferase (referred to hereafter as cap methyltransferase) catalyzes the transfer of a methyl group from AdoMet 1 to the GpppN terminus of RNA to produce m7GpppN-terminated RNA and AdoHcy (1). The Saccharomyces cerevisiae cap methyltransferase is the product of the ABD1 gene (7). ABD1 encodes a 436-amino acid polypeptide. A catalytic domain of Abd1p from residues 110 -426 ...
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