Elizabeth T. Silver

A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s.

The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains coexist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature‐sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi‐ubiquitin chain synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity and function of activating Ly‐49 receptors

Summary: Inhibitory Ly‐49 receptors allow murine natural killer (NK) cells to kill cells with aberrant class I MHC expression while sparing normal cells. This is accomplished by their recognition of specific class I MHC products and prevention of NK‐cell lysis of cells that present a normal repertoire of class I MHC ligands –“the missing self hypothesis”. However, Ly‐49 receptors that lack the cytoplasmic immunoreceptor tyrosine‐based inhibitory motif, which is required for inhibition of killing, have also been described. These receptors were found to stimulate NK killing and are therefore referred to as activating Ly‐49 receptors. Interestingly, the activating receptors have class I MHC‐binding domains that are nearly indistinguishable from those of the inhibiting receptors, and binding to class I MHC has now been demonstrated for three activating receptors. Presently, there is no defined physiological role for activating Ly‐49 receptors. Here we present an overview of current knowledge regarding the diversity, structure and function of activating Ly‐49 receptors with a focus on class I MHC specificity, and we discuss their potential role(s) in natural resistance.

Ly-49P Activates NK-Mediated Lysis by Recognizing H-2Dd 1

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd α1/α2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.

Ly-49W, an Activating Receptor of Nonobese Diabetic Mice With Close Homology to the Inhibitory Receptor Ly-49G, Recognizes H-2D k and H-2D d

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2k or H-2d haplotypes, and Ab-blocking experiments indicated that H-2Dk and Dd are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2k class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.

Allelic Variation in the Ectodomain of the Inhibitory Ly-49G2 Receptor Alters Its Specificity for Allogeneic and Xenogeneic Ligands

The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2Dd and Dk class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes Dd, and not Dk. The specificity for Dk as well as Dd of the wild-type Ly-49GBALB/c allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49GBALB/c allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49GB6. Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.

The Rat RT1-A1c MHC Molecule Is a Xenogeneic Ligand Recognized by the Mouse Activating Ly-49W and Inhibitory Ly-49G Receptors

Mouse Ly-49 receptors are known to recognize xenogeneic ligands from hamster and rat. However, until now, there has been no description of a specific rat xenogeneic ligand for any mouse Ly-49 receptor. In this report, we identify RT1-A1c, a rat classical class I MHC molecule, as a ligand for the Ly-49GBALB/c inhibitory receptor and the closely related activating receptor, Ly-49W. Xenogeneic class I recognition of targets from PVG but not DA strain rats was mapped to the classical region of the RT1c haplotype by using Con A blasts from RT1c/RT1av1 intra-MHC recombinant rats as targets for RNK-16 cells expressing either Ly-49W or Ly-49GBALB/c receptors. Individual expression of class I molecules from PVG and DA rat strains in YB2/0 target cells demonstrate the xenogeneic recognition to be allele specific, because other class I molecules of the RT1c haplotype, RT1-A2c and RT1-U2c, and a classical class I molecule encoded by the RT1av1 haplotype, RT1-Aa, are not recognized by Ly-49W and -GBALB/c. Furthermore, specificity for RT1-Ac can be transferred from Ly-49W to Ly-49P, which is normally unable to recognize RT1-Ac, by substitution of three residues shared by Ly-49W and -GBALB/c but not Ly-49P. These residues are located in the Ly-49 β4–β5 loop, which can determine class I allele specificity in mouse Ly-49 receptor interactions with mouse class I ligands, suggesting that mouse Ly-49 recognition of rat class I molecules follows similar principles of interaction. These findings have implications for xenotransplantation studies and for discerning Ly-49 recognition motifs present in MHC molecules.

Reciprocal Transfer of Class I MHC Allele Specificity between Activating Ly-49P and Ly-49W Receptors by Exchange of β4–β5 Loop Residues

Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2Dd, or H-2Dd and Dk, respectively. In this report, we demonstrate that specificity for H-2Dk can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the β4–β5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2Dk recognition while still preserving H-2Dd recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the β4–β5 loop, in determining class I MHC allele specificity.

Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand

Ly‐49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly‐49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I‐like proteins. Previous studies have provided insight into the nature of Ly‐49A and ‐C interaction with their high‐affinity MHC I ligands, H‐2Dd and Kb, respectively. Unlike Ly‐49C, recognition of MHC I by Ly‐49A is regulated in part by residues within the β4–β5 loop of its ectodomain. Ly‐49A and ‐G are within the same Ly‐49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly‐49G that mediate class I MHC recognition. The Ly‐49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK‐1, which recognizes Ly‐49GB6 and a Ly‐49GB6‐like receptor, Ly‐49Mnonobese diabetic, but not Ly‐49GBALB/c. By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly‐49G allele products, we identified epitopes recognized by the Ly‐49G‐specific antibodies CK‐1 and Cwy‐3, whose epitopes mapped within the β4–β5 loop and the β1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly‐49GB6 ectodomain, Cwy‐3 was unable to block its interaction with MHC I, and CK‐1 significantly inhibited it. The importance of residues within the β4–β5 loop in Ly‐49G recognition demonstrates that its interaction with MHC I is similar to that of Ly‐49A but not Ly‐49C.