Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four α-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.

Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4°C exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1–2 h at 37°C, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.

Euler characteristics and elliptic curves

Let E be a modular elliptic curve over ℚ, without complex multiplication; let p be a prime number where E has good ordinary reduction; and let F∞ be the field obtained by adjoining to ℚ all p-power division points on E. Write G∞ for the Galois group of F∞ over ℚ. Assume that the complex L-series of E over ℚ does not vanish at s = 1. If p ⩾ 5, we make a precise conjecture about the value of the G∞-Euler characteristic of the Selmer group of E over F∞. If one makes a standard conjecture about the behavior of this Selmer group as a module over the Iwasawa algebra, we are able to prove our conjecture. The crucial local calculations in the proof depend on recent joint work of the first author with R. Greenberg.

A sequential dimerization mechanism for erythropoietin receptor activation.

We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and Ser-104 (and possibly Arg-14), is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.