No abstract

Acetylcholine receptors of fish electric organs and mammalian skeletal muscle comprise four structurally homologous glycoprotein subunits in the mole ratio alpha 2 beta gamma delta (refs 1-4). All four subunits have leader sequences and are exposed on both sides of the membrane. From amino acid sequencing, three groups have predicted that each subunit has four hydrophobic alpha-helical transmembranous domains. Because the N-terminus of each subunit is thought to remain on the extracellular surface after cleavage of the leader sequence, this model predicts that the N- and C- termini are both on the extracellular side. An alternative model proposed by two other groups predicts that there is, in addition, a fifth amphipathic transmembranous domain which would place the C-terminus on the cytoplasmic side. Here, using anti-subunit sera and monoclonal antibodies and their reaction with synthetic subunit peptides, we demonstrate that the C-terminus is in fact on the cytoplasmic surface. We also show that, contrary to other predictions, the most hydrophilic sequence on the extracellular domain of alpha-subunits is not the main immunogenic region.

Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the a subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, a subunit, and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide a159-169 was less by a factor of 104, suggesting that the mAbs bind primarily to (6)(7)(8).Analyses of the sequences of cDNAs for acetylcholine receptor subunits have led to several models for the transmembrane organization of the polypeptide chains in all of the subunits and the localization of particular domains, such as the acetylcholine binding site and main immunogenic region, on a subunits (9-13). Antibodies have proven useful in testing the predictions of such studies (14, 15). For example, the COOH termini of all subunits were found on the cytoplasmic surface, and the NH2 termini of all subunits were found to be The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. inaccessible to antibodies in the native receptor (14, 15), results consistent with the idea suggested by their sequence homologies that all of the subunits should have a fundamentally similar structure (10). Demonstration of the cytoplasmic localization of the COOH termini (14,15,47) proved that subunits could not have only the four hydrophobic a-helical transmembrane domains predicted in the models (9-11) but, instead, must have an odd number of transmembrane domains to account for an extracellular location of the NH2 termini and a cytoplasmic location of the COOH termini. This result was consistent with two other models, which proposed a fifth amphipathic a-helical transmembrane domain (12, 13).Noda et al. (16) proposed that the sequence a161-166 composed the main immunogenic region, because this sequence was highly hydrophilic. In fact, mAbs to the main immunogenic region do not bind to synthetic peptides containing this sequence (15,17). Furthermore, we made mAbs to a synthetic peptide containing this sequence and showed that they did not compete for binding to native receptor with mAbs to the main immunogenic region (15). Here we demonstrate that these mAbs bind to the cytoplasmic surface of the receptor.The observation that this part of the a subunit was located on the cytoplasmic surface was unanticipated by all previous models, all of which presumed that it was part of an extracellular domain extending from the NH2 terminus to the first hydrophobic domain, which starts at about a210. Because this sequence is bracketed by two sequences that must be located on the extracellular surface, the only site for N-glycosylation (16) and a cysteine demonstrated to be adjacent to the acetylcholine binding site (18), demonstration of the cytoplasmic location of this sequence rather closely defines the boundaries of two previously unrecognized ...

A method is described for measuring relative binding constants of lysine and analogs of lysine to plasminogen and plasminogen 'kringle' fragments. Plasminogen or kringle fragments adsorbed to lysine-Sepharose are eluted with increasing concentrations of lysine or other ligands, the concentration of ligand required to elute 50% of the protein being taken as a measure of the binding constant. The method is simple and is not dependent on monitoring conformational changes. We confirm earlier reports that the best ligands for the lysine binding sites of plasminogen are w-amino acids containing five or six carbons. We show further that both Glu-plasminogen (the native form with N-terminal glutamic acid) and Lys-plasminogen (a degraded form with N-terminal lysine), as well as the heavy chain fragments, kringle 4 and kringle 1 + 2 + 3, have very similar properties with regard to binding specificity for w-amino acids. For all species optimal binding is observed when the distance between the amino and carboxyl carbon is about 0.68 nm. The binding of ligands is decreased by the presence of polar atoms on the a and p positions of the carbon chain of amino acids.Arginine binds relatively weakly at the lysine site and there does not appear to be a separate arginine binding site in plasminogen.It has been known for over 20 years that amino acids such as 6-aminohexanoic acid have antifibrinolytic properties [l], one of which probably results from inhibition of the binding of plasminogen to fibrin [2]. Subsequent studies have shown that 6-aminohexanoic acid and lysine cause extensive conformational alterations in Glu-plasminogen [3,4] (and references in [4]); Deutsch and Mertz [5] utilized the strong affinity of plasminogen for lysine-Sepharose in the development of a convenient method for purification of plasminogen.

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr∼ 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H‐NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p‐benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysinebinding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl‐region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two‐dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at‐1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H‐NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine‐binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2‐hydroxy‐5‐nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine‐Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine‐binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.