Disulfide Loop Research Articles

Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes.

The staphylococcal enterotoxins (SEs) are homologous proteins related in their capacity for stimulating both T cells and monocytes. To assess the importance of conserved structure and sequence to functional activity, the role of the disulfide loop and adjacent sequence in these toxins was evaluated. Contrary to previous reports, we demonstrate here that the disulfide loop was required for the mitogenic activity of SEA and SEB. While T cell-stimulatory activity was compromised, reduced and alkylated SEs retained major histocompatibility complex class II-binding and monocyte-stimulatory activities, suggesting that their inability to induce T cell proliferation was due to failure to interact with T cell receptor (TCR) rather than with class II molecules. Reduction and alkylation did not affect the far-ultraviolet circular dichroic spectrum of SEA, suggesting that the loss of mitogenic activity was not associated with significant changes in secondary structure. The disulfide linkage imparts considerable stability to these toxins as peptide cleavages within the loop of SEB were not associated with detectable loss of function, although cleavage in the conserved sequence outside the loop of SEA resulted in loss of mitogenic activity. This report thus establishes a functional role for a conserved element in SEs, the disulfide loop, and further indicates that their class II- and TCR-binding activities can be dissociated.

Fibulin is an extracellular matrix and plasma glycoprotein with repeated domain structure.

We have studied the expression of fibulin in cultured fibroblasts and determined its primary structure by cDNA cloning. Our results show that fibulin is a secreted glycoprotein that becomes incorporated into a fibrillar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. In addition, we find that fibulin is present in plasma at a level of 33 +/- 3 micrograms/ml. Sequencing of multiple fibulin cDNAs indicates that a process of alternative splicing results in the expression of three fibulin transcripts. The transcripts encode overlapping polypeptides differing only in carboxy-terminal segments. Common to the three predicted forms of fibulin is a unique 537-amino acid-long cysteine-rich polypeptide and a 29-residue signal peptide. The amino-terminal portion of fibulin contains a repeated element with potential disulfide loop structure resembling that of the complement component anaphylatoxins C3a, C4a, and C5a as well as proteins of the albumin gene family. The bulk of the remaining portion of the molecule is a series of nine EGF-like repeats.

Folding of the reduced form of the thioredoxin from bacteriophage T4

The folding pattern for bacteriophage T4 thioredoxin is similar to that of the oxidized form [Borden, K. L. B., & Richards, F. M. (1990) Biochemistry 29, 3071-3077]. Equilibrium and kinetic studies were carried out by fluorescence and circular dichroism techniques. The same box model proposed for the oxidized form, with four identifiable states, can accommodate most of the data: N----Uc----Ut----It----N, where N is the native state, Uc is the unfolded species with Pro 66 in the cis form, Ut is the unfolded species with Pro 66 in the trans form, and It is a trans-Pro 66 intermediate with a volume comparable to that of N. However, the relative importance of the different components is shifted between the oxidized and reduced proteins. In spite of the small size of the disulfide loop, the Cys 14-Cys 17 bond appears to be important in stabilizing It. The tertiary structure as monitored by near-UV CD and fluorescence indicates that the reduced form is significantly less stable than its oxidized counterpart; however, the two secondary structures, as seen by far-UV CD, are very similar. The intermediate It behaves as though it is cold denaturated at 4 degrees C.

Ca2+ binding properties and Ca2(+)-dependent interactions of the isolated NH2-terminal alpha fragments of human complement proteases C1-r and C1-s.

The NH2-terminal alpha fragments of human complement proteases C1-r and C1-s were obtained by limited proteolysis of the native proteins with trypsin, and isolated. C1-r alpha extended from residues 1 to 208 of C1-r A chain, with at least two cleavage sites within disulfide loops, after lysine 134 and arginine 202. C1-s alpha comprised residues 1-192 of the C1-s A chain, with one cleavage site within a disulfide loop, after arginine 186. C1-r alpha was monomeric either in the presence or absence of Ca2+ but formed Ca2(+)-dependent dimers with native C1-s. C1-s alpha dimerized in the presence of Ca2+ and formed Ca2(+)-dependent tetramers (C1-s alpha-C1-r-C1-r-C1-s alpha) with native C1-r. C1-r alpha and C1-s alpha associated in the presence of Ca2+ to form C1-r alpha-C1-s alpha heterodimers. Equilibrium dialysis studies indicated that each alpha region binds Ca2+ with a dissociation constant ranging from 19 microM (native proteins) to 38 microM (fragments). C1-r alpha, C1-r alpha-C1-s alpha, and the native C1-s-C1-r-C1-r-C1-s tetramer bound 0.9, 1.9, and 4.0 Ca2+ atoms/mol, respectively, whereas dimers C1-s alpha-C1-s alpha and C1-s-C1-s incorporated 2.9 and 3.0 Ca2+ atoms/mol. It is concluded that each alpha region contains one high affinity Ca2+ binding site. This 1:1 stoichiometry is maintained upon heterologous (C1-r-C1-s) interaction, whereas the homologous (C1-s-C1-s) interaction provides one additional binding site.

Molecular Modeling of Residues 38–57 of the β-Subunit of Human Lutropin

Several regions on both the alpha- and beta-subunits of human LH comprise the receptor-binding domain of the hormone. One of these, a disulfide loop peptide containing residues 38-57 on the beta-subunit, also stimulated steroidogenesis in rat Leydig cells. Circular dichroism analysis and a Schiffer-Edmundson helical wheel projection of beta-(38-57) revealed the possibility of an amphipathic alpha-helical structure through its N-terminal region. Secondary structure prediction algorithms do not predict alpha-helix in beta-(38-57), but, rather, suggest a sheet-coil-sheet topology. Homology searches between this peptide and proteins with known structure revealed that the two best matches are with prealbumin-(10-30) and melittin-(1-26). Based on hydrophobic moment calculations, we suggest that beta-(38-57) more closely resembles melittin, a known example of an amphipathic helix. Molecular models were constructed that included an alpha-helix between Pro-39 and Pro-50 producing a hydrophilic face involving Thr-40, Arg-43, and Gin-46. Loop closure was performed either visually or by an incremental minimization procedure, using distance constraints to patch in a disulfide bond. Molecular dynamics at 300, 360, and 1000 K were used to explore the local conformational space, and dynamic structures were minimized. The most reasonable structures were found with the 300 and 360 K simulations, with those at 360 K consistently producing structures with lower conformational energies. In each of these simulations, the N-terminus of the alpha-helix unraveled to form a reverse turn (predicted by the GOR algorithm) which include Cys-38, Pro-39, Thr-40, and Met-41. Simulations at 1000 K produced the most variation in structure, but these were deemed unreasonable. Although not all possible conformations were explored, several models were found that comply with the assumption of an amphipathic helix in the N-terminal half of the peptide.

Local structure in ribonuclease A. Effect of amino acid substitutions on the preferential formation of the native disulfide loop in synthetic peptides corresponding to residues Cys58-Cys72 of bovine pancreatic ribonuclease A

Local structure in ribonuclease A. Effect of amino acid substitutions on the preferential formation of the native disulfide loop in synthetic peptides corresponding to residues Cys58-Cys72 of bovine pancreatic ribonuclease A

Purification and characterization of a large, tryptic fragment of human thyroid peroxidase with high catalytic activity

Thyroid peroxidase (TPO) was purified from human thyroid tissue, obtained at surgery from patients with Graves' disease, by a procedure similar to one that we had previously used for the purification of porcine TPO. The membrane-bound enzyme was solubilized by treatment of the thyroid particulate fraction with trypsin plus detergent. After precipitation with ammonium sulfate, the enzyme was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. Although a high degree of purification was achieved, the finally isolated product was considerably more heterogeneous than the TPO obtained from porcine thyroids. Several pools of active enzyme differing in values for A 412 A 280 and in specific activity were collected. Gel electrophoresis was performed under native, denaturing [sodium dodecyl sulfate (SDS)] and denaturing plus reducing conditions. Native gel electrophoresis indicated that the active enzyme (93 kDa) was heavily contaminated with an inactive 60-kDa fragment, which we were unable to remove by HPLC. The inactive fragment was highly antigenic when tested on immunoblots with an antibody to TPO. The presence of the inactive fragment greatly reduced values for A 412 A 280 in the finally purified human TPO. Two of the pools, with A 412 A 280 values of 0.159 and 0.273, were used for further testing. Catalytic activity was very similar in these two pools when measured on the basis of heme content by several different assays. Moreover, the specific activities of both, based on heme content, were very similar to those observed with a porcine TPO preparation with A 412 A 280 = 0.48 . These findings indicate that the inactive 60-kDa fragment most likely did not contain heme. On SDS-polyacrylamide gel electrophoresis under reducing conditions, the 60-kDa fragment completely disappeared and was replaced by a 36- and a 24-kDa component. Amino terminal sequence information obtained on these components indicated that the 24-kDa component represents the amino terminal portion of the active 93-kDa fragment, whereas the 36-kDa fragment represents the carboxyl terminal portion. A model is proposed suggesting that the 60-kDa fragment was generated by trypsin cleavage of native TPO at two internal sites within a disulfide loop (res ~300 and res 564) and at one further internal site (res 280). In addition, trypsin cleavage is proposed at sites near the amino and carboxyl ends common to both the active 93-kDa and the inactive 60-kDa fragments. According to this model, the inactive 60-kDa fragment is missing that portion of the polypeptide chain extending from res ~300 to res 564. Since the 60-kDa fragment lacks heme, this region of the molecule appears to be important for heme binding and may contain the heme binding site.

Disulfide Bridge Structure of Ascidian Trypsin Inhibitor I: Similarity to Kazal-Type Inhibitors

The primary structures of ascidian trypsin inhibitors (iso-inhibitors I and II) were reported in the preceding paper (Kumazaki, T. et al. (1990) J. Biochem. 107, 409-413). Both of them have eight half-cystines in a molecule composed of 55 amino acid residues with a sequence showing no extensive homology to other known protease inhibitors. To locate the four disulfide bridges in the molecule, native inhibitor I was digested with thermolysin to yield cystine-containing peptides. The peptides were separated from each other by reversed-phase HPLC. A core peptide still containing six closely located half-cystines (e.g. -Cys-Arg-Cys and -Cys-Cys-) was further digested with Streptomyces griseus trypsin for cleavage of the Arg-Cys bond. On the other hand, the Cys-Cys bond was split by applying manual Edman degradation to the core peptide. Amino acid composition analyses of the resulting cystine peptides allowed us to define the whole disulfide bridge structure in the parent molecule. The topological relation between the disulfide loops and the reactive site suggested that the ascidian trypsin inhibitor may be classified as a member of the Kazal-type inhibitor family.

The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily.

The cDNA sequence encoding the bovine fetal protein fetuin is reported. The deduced amino acid sequence is identical with that obtained from amino acid sequencing. The protein is a single chain preceded by a signal sequence. The three N-linked glycosylation sites have been determined. The sequence of fetuin shows over 70% similarity to human alpha 2HS glycoprotein. All of the cysteine residues are conserved in both proteins, suggesting that fetuin has the same arrangement of disulfide loops as alpha 2HS glycoprotein and may also be a member of the cystatin family. Southern blot analysis indicates that a single gene codes for fetuin. No evidence for a separate gene for a bovine alpha 2HS glycoprotein was obtained; thus, fetuin in cattle and alpha 2HS glycoprotein in the human are equivalent proteins.

Evolution of proteins of the cystatin superfamily

We have examined the amino acid sequences of a number of proteins that have been suggested to be related to chicken cystatin, a protein from chicken egg white that inhibits cysteine proteinases. On the basis of statistical analysis, the following proteins were found to be members of the cystatin superfamily: human cystatin A, rat cystatin A(alpha), human cystatin B, rat cystatin B(beta), rice cystatin, human cystatin C, ox colostrum cystatin, human cystatin S, human cystatin SA, human cystatin SN, chicken cystatin, puff adder cystatin, human kininogen, ox kininogen, rat kininogen, rat T-kininogens 1 and 2, human alpha 2HS-glycoprotein, and human histidine-rich glycoprotein. Fibronectin is shown not to be a member of this superfamily, and the c-Ha-ras oncogene protein p21 (Val-12) probably is not a member also. It was convenient to divide members of the superfamily into four types on the basis of the presence of one, two, or three copies of cystatin-like segments and the presence or absence of disulfide bonds. Evolutionary dendrograms were calculated by three methods, and from these we have constructed a scheme depicting the sequence of events in the evolution of these proteins. We suggest that about 1000 million years ago a precursor containing disulfide loops appeared, and that all disulfide-containing cystatins are derived from this. We follow the evolution of the proteins of the superfamily along four main lineages, with special attention to the part that duplication of segments has played in the development of the more complex molecules.

Protein Structural Requirements and Properties of Membrane Binding by γ-Carboxyglutamic Acid-containing Plasma Proteins and Peptides

Abstract The membrane-binding characteristics of a number of modified vitamin K-dependent proteins and peptides showed a general pattern of structural requirements. The amino-terminal peptides from human prothrombin (residues 1-41 and 1-44, 60:40) bovine factor X (residues 1-44), and bovine factor IX (residues 1-42), showed a general requirement for a free amino-terminal group, an intact disulfide, and the tyrosine homologous to Tyr44 of factor X for membrane binding. Consequently, the peptide from factor IX did not bind to membranes. Any of several modifications of the amino terminus, except reaction with trinitrobenzenesulfonic acid, abolished membrane binding by the factor X and prothrombin peptides. Calcium, but not magnesium, protected the amino terminus from chemical modification. The requirement for a free amino terminus was also shown to be true for intact prothrombin fragment 1, factor X, and factor IX. Although aggregation of the peptide-vesicle complexes greatly complicated accurate estimation of equilibrium binding constants, results with the factor X peptide indicated an affinity that was not greatly different from that of the parent protein. The most striking difference shown by the peptides was a requirement for about 10 times as much calcium as the parent proteins. In a manner similar to the parent proteins, the prothrombin and factor X peptides showed a large calcium-dependent quenching of tryptophan fluorescence. This fluorescence quenching in the peptides also required about 10 times the calcium needed by the parent proteins. Thus, the 1-45 region of the vitamin K-dependent proteins contained most of the membrane-binding structure but lacked component(s) needed for high affinity calcium binding. Protein S that was modified by thrombin cleavage at Arg52 and Arg70 showed approximately the same behavior as the amino-terminal 45-residue peptides. That is, it bound to membranes with overall affinity that was similar to native protein S but required high calcium concentrations. These results suggested that the second disulfide loop of protein S (Cys47-Cys72) and prothrombin (Cys48-Cys61) were involved in high affinity calcium binding. Since factor X lacks a homologous disulfide loop, an alternative structure must serve a similar function. A striking property of protein S was dissociation from membranes by high calcium. While this property was shared by all the vitamin K-dependent proteins, protein S showed this most dramatically and supported protein-membrane binding by calcium bridging.

Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids

Microscopic disulfide-exchange rate constants have been measured for the formation and opening of small disulfide loops in reactions between glutathione and peptides containing 2 cysteines. Twelve cysteine-Xm-cysteine peptides have been studied, where X is an amino acid and m is the number of amino acids between the cysteines. Homopolymers of alanine for m equaling 0-5 are evaluated, as well as X1 and X2 series employing glycine, valine, or proline. Equilibrium constants Kc for loop closing are only slightly dependent on the nature of X. Loops with even values of m generally are favored relative to loops with odd values. Kc increases in the rank order X1, X3, X0, X5, X4, and X2. Formation of a disulfide between sequentially adjacent cysteines therefore is not especially difficult. The dependence of Kc on the odd-even nature of m is compared with similar patterns observed both in statistics of disulfide formation in naturally occurring proteins and in theoretical studies of peptide cyclization. The relative equilibrium populations of intramolecular disulfides in peptides containing cysteine-cysteine-cysteine and cysteine-serine-cysteine-serine-cysteine clusters are consistent with predictions based on the values of Kc in the two-cysteine peptides.

Inhibition of epidermal growth factor/transforming growth factor‐α–stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.

Molecular cloning of chromogranin A from rat pheochromocytoma cells.

Chromogranin A (CgA) is the major soluble protein in catecholamine storage vesicles. To gain insight into its function, we isolated CgA clones from a size-selected lambda gt10 rat pheochromocytoma complementary DNA (cDNA) library. The longest cDNA insert identified was 2.2 kb and encoded the entire 462-amino acid open reading frame of rat CgA including an 18-amino acid hydrophobic signal peptide. Comparison of rat CgA with the recently published sequences of bovine CgA and human CgA revealed regions of strong homology at the N-and COOH-termini as well as variant areas predominantly in the middle portion of the molecule. Regions highly conserved and therefore suggestive of functional importance included 1) multiple paired basic residues, which may serve as proteolytic processing signals; 2) a region homologous to porcine pancreastatin, a putative modulator of peptide hormone release; and 3) a short hydrophobic disulfide loop region near the N-terminus that may have a role in the targeting of CgA to secretory vesicles. On the other hand, lack of conservation of the membrane attachment sequence arginine-glycine-aspartic acid argues against its functional importance in CgA. In addition, the presence of a unique polyglutamine region in rat CgA points to a possible messenger RNA (mRNA) splice junction. Northern blot experiments demonstrated the presence of an approximately 2.2 kb rat CgA mRNA in a neuroendocrine distribution (adrenal, brain, pheochromocytoma cells, but not skeletal muscle, heart, or kidney). Southern blot studies were consistent with the presence of a single CgA gene within the rat pheochromocytoma cell genome. Finally, comparison of the present rat pheochromocytoma cDNA clones with those recently obtained from normal rat adrenal gland reveals minor but apparently real differences that suggest CgA microheterogeneity.

A novel concatenated dimer of recombinant bovine somatotropin.

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized in Escherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.

The Arrangement of Disulfide Loops in Human α2-HS Glycoprotein: Similarity to the Disulfide Bridge Structures of Cystatins and Kininogens

Abstract The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Burgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Burgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.

Sequence of the structural gene (rmpM) for the class 4 outer membrane protein of Neisseria meningitidis, homology of the protein to gonococcal protein III and Escherichia coli OmpA, and construction of meningococcal strains that lack class 4 protein

The structural gene (rmpM) of the class 4 outer membrane protein of Neisseria meningitidis has been cloned and sequenced. The derived amino acid sequence reveals a 218-amino-acid protein following a 22-amino-acid signal peptide. The protein shows 94.2% homology with protein III of Neisseria gonorrhoeae and shares its two potential disulfide loops. The protein also shares limited homology with Escherichia coli OmpA. N. gonorrhoeae protein III has been shown to elicit blocking antibodies that prevent the killing of serum-resistant strains by immune sera (P. A. Rice, H. E. Vayo, M. R. Tam, and M. S. Blake, J. Exp. Med. 164:1735-1748, 1986). The very close homology of meningococcal class 4 protein with gonococcal protein III suggests that meningococcal outer membrane preparations containing class 4 protein may similarly stimulate blocking antibodies. In order to investigate the role of the class 4 protein in the pathogenesis of meningococcal infection, we have used an erythromycin resistance gene in developing two meningococcal strains that lack class 4 protein.

The alpha-chain of murine CD8 lacks an invariant Ig-like disulfide bond but contains a unique intrachain loop instead.

The CD8 Ag is a cell surface heterodimer which demarcates predominantly cytotoxic T cells which are restricted by class I MHC Ag. The disulfide bonds within the murine structure were assigned in this study and the alpha-beta-interchain bond involves one or more cysteine residues located in each chain proximal to the plasma membrane or included within it. The location of the intrachain disulfide loop within the CD8 beta-chain confirms its proposed structural homology to an IgV domain but no corresponding disulfide loop is present within the alpha-chain. The invariant IgV disulfide loop has been replaced by a unique, short loop involving an unusual cysteine which is conserved in the CD8 alpha-chains of man, mouse, and rat. Despite its lack of precedent in other Ig-related structures, this unusual disulfide loop can be parsimoniously accommodated into a modified domain which has retained the major features of the Ig structural motif.

A mutant lactogenic hormone binds, but does not activate, the prolactin receptor.

We have generated mutations in mouse placental lactogen II, a hormone in the PRL/GH family that binds to the PRL receptor, to investigate the role of the conserved cysteine residues in hormone function. Disruption of the small C-terminal disulfide loop did not significantly alter hormone activity. Substitution of serine for cysteine-51, which prevents formation of the large disulfide loop, results in a protein equivalent to placental lactogen II in receptor-binding activity; however, this mutant protein is not mitogenic in an assay for lactogenic hormones. These results indicate that PRL receptor occupancy and activation are distinct events.

Antibody-cell interactions: Fc receptors

Jean-Pierre Kinet National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, Maryland 20892 Antibodies are involved in two main functions: the binding to antigen via the antigen-combining site and the mobili- zation of defense mechanisms via another part of the mol- ecule, the Fc region. The attachment of the Fc region to receptors on many cells of the immune system triggers various functions such as phagocytosis, antibody-depen- dent cytotoxicity, and the secretion of potent mediators. Because the Fc receptors (FcRs) mediate these essential functions of the immune defense system, understanding their structure and function is a central problem in immu- nology. Such understanding may also help in designing new therapeutic approaches to allergy and autoimmunity, disorders in which these receptors are involved. In this minireview I discuss the new information about the structures of FcRs that has been obtained by molecu- lar cloning. It is becoming apparent that with one excep- tion (FccRII) the FcRs belong to the immunoglobulin (lg) superfamily, so that both the antibodies and their recep- tors are members of the same general family. However, the structural diversity of these receptors is greater than initially expected. The analysis of homologous receptors from different species is now possible and may help to identify those portions of the molecule that are function- ally important. Special emphasis is given here to the most complex of FcRs: the receptor with high affinity for IgE. One class of Fc receptors, the lg transporters, is not dis- cussed because of space limitations and because their mechanism of action is presumably quite different. Fc Receptors for /gG Distinct forms of FcyRs have been identified both in mice and in humans: a receptor (FcyRI) with high affinity for monomeric IgG, and other receptors (FcyRII and FcyRIII) that have only low affinity for monomeric IgG but that can effectively bind immune complexes by multiple recep- tor-ligand interactions (reviewed in Unkeless et al., 1988). Several mouse and human FcyRs have been cloned. All belong to the lg superfamily and with one exception (FcyRIII-PIG; see below) are integral membrane proteins with a leader peptide, an extracellular portion that is glycosylated, a single transmembrane segment, and a carboxy-terminal cytoplasmic “tail” (see figure). The ami- no-terminal extracellular portion contains two or three lg- related domains that show significant homology with the C2 set of lg domains as defined by A. Williams. These do- mains are formed by disulfide loops of variable length (42-44 residues) that are too short to allow confident pre- diction of their folding relative to a typical lg domain. Despite being members of the same family, FcyRs ex- hibit rich structural diversity. In the mouse, FcyRll is en- coded by two genes a and b. Because of alternative