Homomeric Association Research Articles

Self-assembled structures of ferrocene-l-carnosine conjugates

Ferrocene-l-carnosine conjugates, Fc-l-Carnosine-OEt and Fc2-l-Carnosine-OEt, were synthesized and characterized by X-ray crystallographic analyses. The single-crystal X-ray structure determination of Fc-l-Carnosine-OEt was demonstrated to reveal the formation of antiparallel β-sheet-like 12-membered intermolecularly hydrogen-bonded ring between NH (β-alanine) and CO (β-alanine of another molecule). Furthermore, the self-assembled structure through antiparallel β-sheet-like hydrogen bonding network of β-alanyl moieties and homomeric association of the imidazole moieties through the one-dimensional hydrogen-bonded chain was formed in the crystal packing. The self-assembled structure based on antiparallel β-sheet-like hydrogen bonding between NH (histidine) and CO (the ferrocenoyl moiety of another molecule) was also observed in the crystal packing of Fc2-l-Carnosine-OEt. In the case of Fc2-l-Carnosine-OEt, which has the N-ferrocenoyl imidazole moiety, each molecule is connected through the intermolecular hydrogen bonds of CH⋯N pattern between the ferrocene and the imidazole moieties.

Intertwined Associations in Structures of Homooligomeric Proteins

Intertwined homo-oligomers are complexes comprising identical protein subunits, where small segments or compact protein substructures (domains) are exchanged between the subunits. Using a formal definition of intertwined homo-oligomers, we survey the Protein Data Bank for all such complexes. Results show that intertwining occurs in 13,442 (24%) of all surveyed structures. A majority (∼72%) exchanges one contiguous chain segment of varyinglength. Another ∼10%, exchange structural domains, and the remaining ∼20% display complex intertwining topologies. Smaller proteins are more often intertwined, and intertwining is dominant in solution homodimers. These findings and analyses of various properties of the major category of intertwined complexes, their interfaces and quaternary context, support the physiological role of intertwining in promoting homooligomer stability. Furthermore, the number of different intertwining modes observed in families of related proteins is limited, and likely specific to the protein fold. These findings yield unique insights into the role of intertwining in homomeric association.

174 Self-association prompts proteins for new function: The role of altered dynamic properties

Local backbone flexibility along the chain was analyzed in pairs of monomer and homodimeric protein structures with identical sequence. We identified pairs, where highly flexible regions (corresponding to prominent peaks in rmsd’s or crystallographic B-factor values) clearly ‘migrate’ to a new location along the polypeptide in the homodimer. We present several systems, where the new flexible region can be linked to the recruitment of a 3rd binding partner, and where this recruitment is not reported for the corresponding monomer. Conformational sampling of these systems over microsecond timescales further confirms the flexibility migration phenomenon. Compelling evidence is provided that altered backbone flexibility in the homodimer state enables specific recruitment of the heterologous binding partner through the process of conformational selection. Our findings lend support to conformational selection as a general mechanism for protein–ligand binding and highlight the regulatory allosteric role played by the homomeric association event itself. There are many examples of allosteric behavior in homo-oligomers, but this is the first time that homomer association is shown to be the binding event that alters binding properties elsewhere in the protein.

TESTS OF INTEGRIN TRANSMEMBRANE DOMAIN HOMO‐OLIGOMERIZATION DURING INTEGRIN LIGAND BINDING AND SIGNALING

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside‐out and outside‐in signaling, the role of TM homomeric association remains elusive. Formation of TM homo‐oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo‐oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding, or during inside‐out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide‐bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo‐oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside‐in signaling. Therefore, integrin TM homo‐oligomerization is not required for integrin activation, ligand binding or signaling.

Tests of Integrin Transmembrane Domain Homo-oligomerization during Integrin Ligand Binding and Signaling

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.

Intracellular localization and interaction of mRNA binding proteins as detected by FRET

BackgroundA number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.ResultsAll ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1.ConclusionsBiochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.

Detection of Homo- or Hetero-Association of Doks by Fluorescence Resonance Energy Transfer in Living Cells

The Dok proteins represent a family of adaptor proteins serving as common substrates for protein tyrosine kinases and play an important role in regulating signal transduction in multiple cell functions. Dimerization of Dok proteins may represent a powerful and flexible regulatory mechanism that can achieve a variety of consequences. This study aims to detect the homo- or hetero-association of Doks in living cells. The transfection of CFP or YFP fusion protein constructs was carried out using lipofectamine 2000. FRET Measurements were performed using three-channel microscopy and Spectroscopy. By using fluorescence resonance energy transfer technology, we demonstrated, for the first time to our knowledge, that Dok5 and Dok1 could form homomeric and heteromeric associations in living cells. Moreover, pleckstrin homology (PH) domain was found to be essential for homomeric associations of Dok5, while PH domain and phosphotyrosine binding domain were found to be crucial for homomeric associations of Dok1 or heteromeric associations between Dok1 and Dok5. The mechanisms underlying Doks' association may benefit the further understanding of the important role of Dok proteins in regulating signal transduction activated by tyrosine kinases.

A Dimerization Hierarchy in the Transmembrane Domains of the HER Receptor Family

Bitopic membrane proteins offer an opportunity for studying transmembrane domain interactions without the structural complexity inherent to multitopic integral membrane proteins. To date, only homomeric associations have been extensively studied quantitatively. Here we propose to assess the thermodynamics of heteromeric associations, which opens the way to investigating specificity and selectivity. A very interesting system of biological relevance with single transmembrane domains possibly involved in interactions with different partners is the EGFR receptor family. The four members, all tyrosine kinase receptors, are involved in an interaction network that potentially leads to a complete set of homo- and heterodimers, ideally suited to such a study. Furthermore, the transmembrane domains of these receptors have been previously implicated in their function in the past by mutations in the transmembrane domain leading to constitutive activation. We demonstrate, using a fluorescence-based measurement of interaction energies, a hierarchy of transmembrane domain interactions ranging from a noninteractive pair to strong dimerization. We propose a structural model based on the crystal structure of the EGFR dimer, to show how the dimeric structure favors these interactions. The correlation we observe between transmembrane domain and whole receptor interaction hierarchies opens a new perspective, suggesting a role for transmembrane receptor domains in the modulation of receptor signaling.

Factors Influencing the Homomeric and Heteromeric Association of Platelet Integrin Transmembrane Domains.

Integrin are α/β heterodimers that mediate an array of cell-cell and cell-matrix interactions including platelet adhesion and aggregation. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations that are regulated by transmembrane (TM) domain interactions: when integrins are inactive, the TM domains of their α and β subunits interact; the domains separate when integrins assume their active conformation. Platelets express five α subunits (α2, αIIb, αv, α5, and α6) and two β subunits (β1 and β3) that combine to form five adhesions receptors. Previously, we observed that the αIIb and β3 TM domains undergo both heteromeric and homomeric interactions and there is overlap of the interfaces that mediates these interactions. Less is known about the other platelet integrins. To study their interactions, we used the TOXCAT assay. In TOXCAT, a chimeric protein consisting of an N-terminal ToxR' DNA binding domain, a C-terminal maltose-binding protein domain, and an interposed TM domain is expressed in the E. coli inner membrane. TM domain-mediated dimerization of the chimeric protein drives the transcriptional activation of a chloramphenicol acetyl transferase (CAT) reporter gene. To enable TOXCAT to measure heteromeric as well as homomeric interactions, we introduced an R68K mutation into the ToxR' DNA-binding region, thereby preventing CAT synthesis without affecting protein expression. Thus, when both wild-type and disabled ToxR' are concurrently expressed from the same plasmid, disabled ToxR' exerts a dominant-negative effect on CAT synthesis. Using this assay, we found that the interaction of platelet integrin TM domains correlated with the presence of a small residue (sr)-xxx-small residue motif (sr-x3-sr) where x = any residue: α2, αIIb and β1, each of which contains a Gx3G motif, had the strongest tendency to undergo specific homomeric association, whereas α2+β1 and αIIb+β3 had the strongest tendency to form heterodimers. In the TM domains of αv, α5 and β3, one or more of the glycines in sr-x3-sr is replaced by Ser or Ala; as a result, homomeric interactions involving these subunits are substantially weaker. Moreover, mutating each of the small residues in sr-x3-sr to Leu precluded the formation of TM domain oligomers, emphasizing the importance of the sr-x3-sr motif. The dominant-negative TOXCAT assay was also used to screen for inactivating αIIbβ3 and αvβ3 mutations. By introducing random mutations into the β3 TM domain and selecting mutants based on a reduction in CAT synthesis, we identified mutations that enhanced heteromeric αIIbβ3 and αvβ3 association. It is noteworthy that mutations that enhanced the interaction of β3 with αIib were present along the face of the β3 TM helix containing the sr-x3-sr motif. By contrast, mutations enhancing αvβ3 association were distributed throughout the β3 TM helix and didn't cluster around the sr-x3-sr motif. In summary, we have demonstrated that the TM domains of platelet integrin subunits, in addition to αIIb and β3, undergo specific heteromeric and homomeric interactions, suggesting that TM domain interactions may regulate the function of the integrins containing these subunits. Further, our results indicate that sr-x3-sr motifs play an essential role in the oligomerization of these subunits, suggesting that these motifs play a central role in regulating integrin function.

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins α Mβ 2 plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between β 2 subunits is higher than α M subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmembrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between α M or β 2 subunits.

Activation of Platelet α Iibβ 3 by Exogenous Peptides Corresponding to the Transmembrane Domains of α Iib and β 3.

The platelet fibrinogen receptor α IIbβ 3 exists in an equilibrium between inactive and active conformations. In its inactive conformation, the transmembrane (TM) domains of α IIb and β 3 interact, but they separate when α IIbβ 3 assumes its active conformation. Peptides corresponding to the α IIb TM domain form homodimers in vitro and in bacterial membranes and the interface that mediates this interaction overlaps with the interface that mediates the heteromeric association of the α IIb TM domain with that of β 3. Because the homomeric association of α IIb TM domain is relatively strong, we expected that peptides spanning the α IIb TM domain might activate α IIbβ 3 by binding to the α IIb TM domain, thereby disrupting the α IIb/β 3 TM domain heterodimer. We synthesized a 26 residue peptide corresponding to the α IIb TM domain, flanked by pairs of lysine residues to increase its solubility in water, and assessed its ability to activate washed or gel-filtered platelets in an aggregometer. Addition of 3 μM peptide induced platelet aggregation after a short lag, but unlike aggregation stimulated by ADP, peptide-induced aggregation was not preceded by platelet shape change. Nonetheless, like ADP-induced aggregation, peptide-induced aggregation was inhibited by EDTA and the α IIbβ 3-specific monoclonal antibody A2A9. On the other hand, pre-incubating platelets with PGE1 or apyrase caused only a small decrease in the rate and extent of peptide-induced aggregation, suggesting that the peptide induced aggregation by binding directly to α IIbβ 3. A peptide corresponding the β 3 TM domain behaved in an identical manner, whereas an unrelated peptide, the model TM domain MS-1, had no effect. Similarly, there was little response to an α IIb TM peptide in which glycines 972 and 976, the first and last residues of a GxxxG motif, were changed to leucine. Confirming that the α IIb TM peptide binds to α IIbβ 3, we found that a FITC-labeled α IIb TM peptide co-eluted with purified α IIbβ 3 during size exclusion gel filtration. To determine whether the latter interaction involved the α IIb TM domain, we used the TOXCAT assay. In TOXCAT, the α IIb TM domain-mediated dimerization of a fusion protein containing the α IIb TM domain interposed between maltose binding protein and the ToxR' transcriptional activation domain in the inner membrane of E. coli drives the activation of the chloramphenicol acetyl transferase (CAT) gene. We found that adding the α IIb TM peptide to the bacterial growth medium consistently decreased CAT synthesis, implying that its presence impaired α IIb-mediated fusion protein dimerization. These results provide evidence for the presence of an α IIb/β 3 TM domain heterodimer in unstimulated human platelets and suggest that the GxxxG motif in the α IIb TM domain is involved in its formation. Because the homomeric interaction of α IIb and β 3 TM domains is substantially stronger than their heteromeric interaction, our data are also consistent with the hypothesis that the TM peptides disrupt the heterodimer which functions to maintain α IIbβ 3 in an inactive state. Lastly, the data suggest that stabilizing the α IIb/β 3 TM heterodimer may represent a new approach for the development of novel anti-platelet agents.

A push-pull mechanism for regulating integrin function.

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.

Heteromeric and Homomeric Transmembrane Domain Associations Regulate αIIbβ3 Function.

The integrin αIIbβ3 resides on the platelet surface in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor αIIbβ3 in the platelet membrane. Accordingly, the αIIb and β3 transmembrane (TM) domains, located near the ends of the stalks, are in proximity when αIIbβ3 is inactive and separate upon αIIbβ3 activation. Peptides corresponding to these domains undergo both homomeric and heteromeric interactions in biological membranes. Thus, it is possible that the shift between inactive and active αIIbβ3 conformations is accompanied by a shift from heteromeric to homomeric αIIb and β3 TM domain interactions. Indeed, we reported that introducing Asn, a residue known to strengthen homomeric TM helix interactions, into the β3 TM domain shifts αIIbβ3 to an active state. As a further test of this model of αIIbβ3 regulation, we studied the effects of mutations of the αIIb TM domain. First, we placed Asn at 10 consecutive positions in the αIIb TM domain, extending from residues V969 to L978, and co-expressed each mutant with WT β3 in CHO cells. Only one of the mutants, G972N, was constitutively active, binding ~ 8-fold more fibrinogen than WT αIIbβ3. Moreover, G972N was expressed in non-uniform patches on the CHO cell surface, consistent with the formation of αIIbβ3 clusters. G972 is the first residue of a GxxxG motif that is essential for dimerization of the αIIb TM domain. Using the TOXCAT assay to assess TM domain dimerization, we observed that G972N results in a 55% decrease in TOXCAT activity. This implies that the effect of G972N on the αIIbβ3 activation state was not a result of increased homo-dimerization of αIIb, but it is more likely that the mutation disrupted its heteromeric interaction with β3. To test this suggestion, we introduced mutations known to disrupt αIIb homo-dimerization (G972L, G976A, and G976L) into αIIbβ3 and measured their effect on αIIbβ3 function. Like G972N, each mutation induced constitutive αIIbβ3 activation and clustering. Lastly, we measured the effect of L980A, a mutation in the αIIb TM domain that unlike G972N, results in a 2.5-fold increase in TOXCAT activity. CHO cells expressing L980A constitutively bound ~ 6.5-fold more fibrinogen than did cells expressing WT αIIbβ3. Taken together, our results suggest a mechanism for αIIbβ3 regulation that involves both the heteromeric and homomeric association of the αIIb and β3 TM domains. Any process that destabilizes the heteromeric association of the αIIb and β3 TM domains would be expected to allow dissociation of these domains with concomitant αIIbβ3 activation. Hence, mutations that disrupt the heteromeric αIIb/β3 TM domain interface “push” αIIbβ3 toward activation. Conversely, intermolecular interactions that either require separation of the αIIb and β3 TM domains or are more favorable when they dissociate, such as homo-oligomerization of the αIIb and β3 TM domains, will “pull” the equilibrium toward the activated state.

Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations.

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

Identification of a Region within the ErbB2/HER2 Intracellular Domain That Is Necessary for Ligand-independent Association

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads to erbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966-968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.

Cbln3, a Novel Member of the Precerebellin Family that Binds Specifically to Cbln1

Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide cerebellin. Although cerebellin has properties of a conventional neuropeptide, its function is controversial because Cbln1 has structural features characteristic of circulating atypical collagens. Cbln1 is related to the three subunits of the complement C1q complex. Therefore, we hypothesized that Cbln1 participated in analogous heteromeric complexes with precerebellin-related proteins. Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a cDNA encoding a novel Cbln1-related protein, designated Cbln3. The gene encoding cbln3 had the same intron-exon structure as cbln1 but mapped to a different mouse chromosome (14). The deduced amino acid sequence of Cbln3 was 55% identical to Cbln1 and also contained a C1q signature domain and signal sequence for secretion. In addition to binding avidly to Cbln3, Cbln1 also formed homomeric complexes. In contrast, Cbln3 homomeric association was weak. These interactions exhibited specificity because C1qB bound to neither Cbln1 nor Cbln3. Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear nucleus in which it was detected in granule neurons. Because Cbln1 and Cbln3 are coexpressed in the brain and interact avidly, they may function as a secreted heteromeric complex in vivo.

Heteromeric and homomeric interactions correlate with signaling activity and functional cooperativity of Smad3 and Smad4/DPC4.

Homologs of Drosophila Mad function as downstream mediators of the receptors for transforming growth factor beta (TGF-beta)-related factors. Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGF-beta activities and are essential mediators of the natural TGF-beta response. We now show that Smad3 and Smad4 associate in homomeric and heteromeric interactions, as assessed by yeast two-hybrid and coimmunoprecipitation analyses. Heteromeric interactions are mediated through the conserved C-terminal domains of Smad3 and Smad4. In Smad3, the homomeric interaction is mediated by the same domain. In contrast, the homomeric association of Smad4 requires both the N-terminal domain and the C-terminal domain, which by itself does not homomerize. Mutations that have been associated with impaired Mad activity in Drosophila or decreased tumor suppressor activity of Smad4/DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-terminal domains, impair the ability of Smad3 and Smad4 to undergo homo- and heteromeric associations. Analyses of the biological activity of Smad3 and Smad4 and their mutants show that full signaling activity correlates with their ability to undergo efficient homo- and heteromeric interactions. Mutations that interfere with these interactions result in decreased signaling activity. Finally, we evaluated the ability of Smad3 or Smad4 to induce transcriptional activation in yeast. These results correlate the ability of individual Smads to homomerize with transcriptional activation and additionally with their biological activity in mammalian cells.

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.