The Lnx family proteins function as molecular scaffolds for Numb family proteins.

Drosophila Numb functions as a cell fate determinant during neurogenesis. We isolated a novel mammalian protein, Lnx2, which interacts with mammalian Numb and Numblike. Lnx2 and the related Lnx1 are multimodular proteins that bind to Numb via their NPXY motifs. In addition, Lnx proteins form oligomers either via their PDZ domains binding to PDZ-binding consensus motifs located in their C-termini or by homophilic oligomerization of their RING fingers. Therefore, Lnx proteins may form large networks by homomeric binding. In situ hybridization analysis revealed complementary patterns of Lnx1 and Lnx2 expression in developing and adult brain, although in several structures they are present in the same cell populations. Moreover, their expression patterns overlap with those of the Numb proteins. Oligomerization of Lnx2 and Numb binding occurs simultaneously. Therefore, our findings suggest that Lnx proteins may serve as molecular scaffolds that localize unrelated, interacting proteins, such as Numb, to specific subcellular sites.

Neuronal interleukin-16 (NIL-16): a dual function PDZ domain protein.

Interleukin (IL)-16 is a proinflammatory cytokine that has attracted widespread attention because of its ability to block HIV replication. We describe the identification and characterization of a large neuronal IL-16 precursor, NIL-16. The N-terminal half of NIL-16 constitutes a novel PDZ domain protein sequence, whereas the C terminus is identical with splenocyte-derived mouse pro-IL-16. IL-16 has been characterized only in the immune system, and the identification of NIL-16 marks a previously unsuspected connection between the immune and the nervous systems. NIL-16 is a cytosolic protein that is detected only in neurons of the cerebellum and the hippocampus. The N-terminal portion of NIL-16 interacts selectively with a variety of neuronal ion channels, which is similar to the function of many other PDZ domain proteins that serve as intracellular scaffolding proteins. Among the NIL-16-interacting proteins is the class C alpha1 subunit of a mouse brain calcium channel (mbC alpha1). The C terminus of NIL-16 can be processed by caspase-3, resulting in the release of secreted IL-16. Furthermore, in cultured cerebellar granule neurons undergoing apoptosis, NIL-16 proteolysis parallels caspase-3 activation. Cerebellar granule neurons express the IL-16 receptor CD4. Exposure of these cells to IL-16 induces expression of the immediate-early gene, c-fos, via a signaling pathway that involves tyrosine phosphorylation. This suggests that IL-16 provides an autocrine function in the brain. Therefore, we hypothesize that NIL-16 is a dual function protein in the nervous system that serves as a secreted signaling molecule as well as a scaffolding protein.

Bcl-xS and Bad potentiate the death suppressing activities of Bcl-xL, Bcl-2, and A1 in yeast.

Members of the Bcl-2 family can be grouped into three classes based upon their effects on cell death. The first class suppresses death and includes Bcl-2. A second group, which includes Bax, is lethal, whereas a third class, including Bcl-xS, potentiates killing, although the members are not lethal by themselves. The proteins in the last class are proposed to exert their activity by binding to anti-apoptotic family members, thereby making the cell more susceptible to killing by another agent. To test this hypothesis, an inducible yeast expression system is reported that permits the functional analysis of three Bcl-2 family members. In yeast, Bax is lethal, and this activity is suppressed by Bcl-xL, Bcl-2, and A1. Co-expression of Bcl-xS did not diminish the ability of any of the anti-apoptotic members to antagonize Bax. Rather, co-expression of Bcl-xS potentiated the anti-death activity of all three proteins. This effect was not the result of changes in either the levels or integrity of Bax or anti-apoptotic proteins. Thus, Bcl-xS can bind to anti-apoptotic family members, but this association does not result in loss of biological activity. Therefore, Bcl-xS may act downstream of Bax and in a pathway that is conserved in yeast.

CIPP, a novel multivalent PDZ domain protein, selectively interacts with Kir4.0 family members, NMDA receptor subunits, neurexins, and neuroligins.

We report a novel multivalent PDZ domain protein, CIPP (for channel-interacting PDZ domain protein), which is expressed exclusively in brain and kidney. Within the brain, the highest CIPP mRNA levels were found in neurons of the cerebellum, inferior colliculus, vestibular nucleus, facial nucleus, and thalamus. Furthermore, we identified the inward rectifier K+ (Kir) channel, Kir4.1 (also called "Kir1.2"), as a cellular CIPP ligand. Among several other Kir channels tested, only the closely related Kir4.2 (or "Kir1.3") also interacted with CIPP. In addition, specific PDZ domains within CIPP associated selectively with the C-termini of N-methyl-D-aspartate subtypes of glutamate receptors, as well as neurexins and neuroligins, cell surface molecules enriched in synaptic membranes. Thus, CIPP may serve as a scaffold that brings structurally diverse but functionally connected proteins into close proximity at the synapse. The functional consequences of CIPP expression on Kir4.1 channels were studied using whole-cell voltage clamp techniques in Kir4.1 transfected COS-7 cells. On average, Kir4.1 current densities were doubled by cotransfection with CIPP.

c-Maf interacts with c-Myb to regulate transcription of an early myeloid gene during differentiation.

The MafB transcriptional activator plays a pivotal role in regulating lineage-specific gene expression during hematopoiesis by repressing Ets-1-mediated transcription of key erythroid-specific genes in myeloid cells. To determine the effects of Maf family proteins on the transactivation of myeloid-specific genes in myeloid cells, we tested the ability of c-Maf to influence Ets-1- and c-Myb-dependent CD13/APN transcription. Expression of c-Maf in human immature myeloblastic cells inhibited CD13/APN-driven reporter gene activity (85 to 95% reduction) and required the binding of both c-Myb and Ets, but not Maf, to the promoter fragment. c-Maf's inhibition of CD13/APN expression correlates with its ability to physically associate with c-Myb. While c-Maf mRNA and protein levels remain constant during myeloid differentiation, formation of inhibitory Myb-Maf complexes was developmentally regulated, with their levels being highest in immature myeloid cell lines and markedly decreased in cell lines representing later developmental stages. This pattern matched that of CD13/APN reporter gene expression, indicating that Maf modulation of c-Myb activity may be an important mechanism for the control of gene transcription during hematopoietic cell development.

Modulation of cell death in yeast by the Bcl-2 family of proteins.

Bcl-2 family members are regulators of cell death. The precise biochemical properties of these proteins are unclear although intrafamily protein-protein association is thought to be involved. To elucidate structure-activity relationships among Bcl-2 proteins and identify the pathways in which they act, an inducible death suppressor assay was developed in yeast. Only Bax and Bak killed yeast via a process that did not require interleukin-1beta-converting enzyme-like proteases. Bax/Bak lethality was suppressed by coexpression of Bcl-2 family members that are anti-apoptotic in vertebrates, namely Bcl-xL, Bcl-2, Mcl-1, and A1. Furthermore, Bcl-xL and Bcl-2 suppressed Bax toxicity by distinct mechanisms in yeast. Bad, Bcl-xS, and Ced-9 lacked suppressor activity. These inactive proteins bound to anti-apoptotic members of the Bcl-2 family but not to Bax or Bak. In contrast, most Bcl-2 family proteins that attenuated death bound to Bax and Bak. However, two mutants of Bcl-xL suppressed Bax-induced cell death while having no Bax binding activity. Therefore, Bcl-xL functions independently of Bax binding, perhaps by interacting with a common target or promoting a pathway that antagonizes Bax. Thus, the pathways downstream of Bax and Bcl-xL may be conserved between vertebrates and yeast. This suppressor assay could be used to isolate components of these pathways.

USF2/FIP associates with the b-Zip transcription factor, c-Maf, via its bHLH domain and inhibits c-Maf DNA binding activity.

In screening for proteins that interact with the basic zipper (bZip) transcription factor, c-Maf, we isolated USF2/FIP. USF2 is a member of the bHLH-Zip protein family, possessing a basic (b) DNA binding region, a helix-loop-helix (HLH) motif, and a leucine zipper (Zip) structure. Mutants of USF2 that lacked a Zip formed heterodimers with c-Maf, but did not homodimerize. Deletion of the USF2 basic region or mutation of its helices abrogated its binding to c-Maf, but had no effect on homodimerization. A functional c-Maf bZip motif was necessary for both homodimerization and heterodimerization with USF2. These data suggest a tetrameric configuration for Maf-USF2 complexes. In the presence of USF2, the DNA binding activity of c-Maf was markedly reduced. Therefore, USF2 and c-Maf may interact to regulate gene expression.

Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion protein.

The cellular prion protein (PrP) binds to the C-terminus of Bcl-2 but not Bax. Therefore, we examined whether the C-terminus of Bcl-2 was important for other homomeric and heteromeric protein-protein interactions. Using the yeast two hybrid system and co-immunoprecipitation, three sites of homomeric interactions were identified within Bcl-2. The carboxy terminal 37 amino acids selectively homodimerized. Two additional regions of Bcl-2 (residues 1-129 and 126-200) interacted with each other, but not themselves permitting both intra- and intermolecular association. In addition, we analyzed heteromeric interactions of Bcl-2 with PrP and two Bcl-2 related proteins, Bax and A1. The domain requirements for binding of those three proteins to Bcl-2 were different from one another. Bax binding required almost the entire Bcl-2 molecule, while A1 bound to the amino terminal region (residues 1-82). PrP associated with the carboxy terminus of Bcl-2 (amino acids 200-236). These data suggest configurational models for Bcl-2 containing complexes. First, Bcl-2 may exist as both heterodimers and heteromultimers. Second, molecules such as Bax and A1 may serve to cap chains of Bcl-2 homodimers by interacting with dimerization domains in the extramembrane region. PrP may disrupt chains of Bcl-2 molecules at the homomeric association site in the transmembrane region.

The cellular prion protein (PrP) selectively binds to Bcl-2 in the yeast two-hybrid system.

Bcl-2 can rescue neurons from death and might, therefore, exert its action by associating with neuron-specific proteins. Using LexA-Bcl-2 as bait, we find that the cellular prion protein (PrP) interacts with Bcl-2, but not Bax, in the yeast two-hybrid system. Since the PrP gene has been implicated in neurodegenerative disorders, this preliminary observation suggests a potential pathogenic mechanism for these conditions.

The maf proto-oncogene stimulates transcription from multiple sites in a promoter that directs Purkinje neuron-specific gene expression.

L7 is expressed in all adult cerebellar Purkinje cells, although during development it appears in a stereotyped spatial and temporal pattern that is manifested as parasagittal domains of neurons. Mutations of the L7 promoter in transgenic mice have established that these domains represent functional compartments of Purkinje neurons. Therefore, it is hoped that by defining the transcriptional control of the L7 gene insights into the mechanisms that control functional fate and organization in the nervous system can be gained. Fragments of the L7 promoter were introduced into a selectable reporter gene in Saccharomyces cerevisiae, and these strains were used to select for cerebellar cDNAs encoding proteins that can bind to, and activate transcription from, these elements. This assay identified the c-Maf proto-oncogene as activating transcription from two sites in the L7 promoter. We did a functional domain analysis of vertebrate c-Maf based upon transcriptional activation in S. cerevisiae and showed the requirement for a transactivation domain, leucine zipper, and DNA-binding region in c-Maf. The c-Maf interaction site was mapped to the sequence G/TGG/CNG/TNCT CAGNN in the L7 promoter, which represents an atypical 12-O-tetradecanoate-13-acetate-responsive element-type Maf-responsive element. However, neither Fos nor Jun, either alone or in combination with each other or c-Maf, altered transcription from this element. In contrast, a Maf-related protein, Nrl, completely mimicked c-Maf actions. These data suggest that Maf may interact with additional basic-zipper proteins that determine a subtype of Maf-responsive element binding.

IFN-gamma receptor-Ig fusion proteins. Half-life, immunogenicity, and in vivo activity.

Two mouse IFN-gamma receptor (MoIFN gamma R)-Ig fusion proteins, which were constructed for the purpose of creating new efficient mouse IFN-gamma (MoIFN-gamma) inhibitor molecules, were studied in vivo to determine their plasma half-life and immunogenicity, and to show their biologic activity. The hybrid proteins show 40-h blood persistency. They do not provoke an antibody response when injected into mice, and they are biologically active in vivo, as demonstrated by the prevention of streptozotocin-induced diabetes. The two fusion proteins are efficient MoIFN-gamma antagonists and can be used in mouse models of human diseases to investigate the role of MoIFN gamma in these pathologic states.

Construction, purification, and characterization of new interferon gamma (IFN gamma) inhibitor proteins. Three IFN gamma receptor-immunoglobulin hybrid molecules.

Three efficient mouse interferon gamma (MoIFN gamma) inhibitors were constructed, which consist of the MoIFN gamma receptor (MoIFN gamma R) extracellular portion and constant domains of immunoglobulin (Ig) molecules. These are: 1) the constant domain of the mouse kappa chain, 2) the hinge region and the constant domains 2 and 3 of the mouse gamma 2a chain, and 3) the hinge region and the constant domains 2 and 3 of the human gamma 3 chain. The hybrid molecules were expressed in the mouse myeloma cell line J558L and recovered from the supernatants of cell cultures in one purification step. The proteins MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 form homodimers, whereas MoIFN gamma R-M kappa is a monomer. All three constructs inhibit the binding of radiolabeled MoIFN gamma to its receptor on L1210 cells. They are biologically active in vitro, neutralizing the action of MoIFN gamma in an antiviral activity assay. The fusions of Ig regions to the soluble MoIFN gamma R do not decrease the affinity of the binding site for the ligand. MoIFN gamma R-M kappa has about the same affinity as the soluble MoIFN gamma R and the cell surface receptor of L1210 cells in situ, which are also monomers, whereas the dimers MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 display a 5-10-fold higher affinity for MoIFN gamma than the monomeric molecules. This is best documented in the efficacy of the inhibitors to antagonize the antiviral activity of MoIFN gamma, as the dimeric constructs are about 10 times more active than MoIFN gamma R-M kappa and the soluble MoIFN gamma R. The hybrid constructs can be used as high efficiency MoIFN gamma inhibitors in mouse models of several pathological states in humans, where IFN gamma is thought to play a disease-promoting role.