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PubMed2004

A single residue modulates tyrosine dephosphorylation, oligomerization, and nuclear accumulation of stat transcription factors.

The NH(2) terminus of Stat proteins forms a versatile protein interaction domain that is believed to use discrete surfaces to mediate oligomerization and tyrosine dephosphorylation of Stat dimers. Here we show for Stat1 and Stat5a/b that these interfaces overlap and need to be reassigned to an unrelated region of the N-domain. Unexpectedly, our study showed for Stat1 that defective oligomerization of DNA-bound dimers was associated with prolonged interferon-induced nuclear accumulation. This uncoupling of DNA binding and nuclear retention was explained by the concomitant dephosphorylation deficiency that both Stat1 and Stat5a/b have in common and that for Stat1 was due to defective dephosphorylation by the phosphatase TC45. Furthermore, diminished N-domain-mediated oligomerization affected transcriptional activation by both Stat1 and Stat5a/b in a promoter-specific manner. DNA binding analysis indicated that oligomerization of Stats on DNA may be common, irrespective of the presence of multiple canonical binding sites. Accordingly, also transcription from promoters with only a single discernable gamma-activated sequence site was negatively effected by reduced tetramerization. Thus, these results indicate that defective oligomerization cannot generally be compensated for by enhanced tyrosine phosphorylation and prolonged nuclear accumulation. In addition, these data clarify the role of DNA binding in nuclear retention of Stat1.

Cell Nucleus↗
PubMed2004

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis.

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.

Acrylamide↗
PubMed2005

Constitutively active homo-oligomeric angiotensin II type 2 receptor induces cell signaling independent of receptor conformation and ligand stimulation.

Members of the G-protein-coupled receptor superfamily (GPCRs) undergo homo- and/or hetero-oligomerization to induce cell signaling. Although some of these show constitutive activation, it is not clear how such GPCRs undergo homo-oligomerization with transmembrane helix movement. We previously reported that angiotensin II (Ang II) type 2 (AT(2)) receptor, a GPCR, showed constitutive activation and induced apoptosis independent of its ligand, Ang II. In the present study, we analyzed the translocation and oligomerization of the AT(2) receptor with transmembrane movement when the receptor induces cell signaling. Constitutively active homo-oligomerization, which was due to disulfide bonding between Cys(35) in one AT(2) receptor and Cys(290) in another AT(2) receptor, was localized in the cell membrane without Ang II stimulation and induced apoptosis without changes in receptor conformation. These results provide the direct evidence that the constitutively active homo-oligomeric GPCRs by intermolecular interaction in two extracellular loops is translocated to the cell membrane and induces cell signaling independent of receptor conformation and ligand stimulation.

Animals↗
PubMed2006

Nonvisual arrestin oligomerization and cellular localization are regulated by inositol hexakisphosphate binding.

Interactions between arrestins and phosphoinositides have been reported to regulate multiple membrane-associated signaling and trafficking events including clathrin-mediated endocytosis and light adaptation in Drosophila. Arrestins have been proposed to have nuclear and cytosolic functions as well, although the ligand dependence of these functions has not been investigated. Here we characterize the structural, molecular, and cellular interactions between arrestin-2 and inositol hexakisphosphate (inositol 1,2,3,4,5,6-hexakisphosphate (IP(6))). The crystal structure of the arrestin-2.IP(6) complex was solved to 2.9 A with crystal lattice contacts suggesting two sites on a protein monomer mediating IP(6) binding. Mutagenesis coupled to isothermal titration calorimetry and tritiated IP(6) binding assays confirmed two-site binding with a low affinity IP(6)-binding site in the N-domain and a high affinity site in the C-domain. Native gel electrophoresis, gel filtration, and analytical ultracentrifugation demonstrated the ability of IP(6) to promote arrestin-2 oligomerization via the two crystallographically defined ligand-binding locations. In addition, analysis in mammalian cells revealed that arrestin-2 not only undergoes homo-oligomerization, but it can also hetero-oligomerize with arrestin-3 in a manner that depends on IP(6)-binding sites. Mutation of either IP(6)-binding site in arrestin-2 disrupted oligomerization while interactions with known binding partners including clathrin, AP-2, and ERK2 were maintained. Subcellular localization studies showed that arrestin-2 oligomers are primarily cytoplasmic, whereas arrestin-2 monomers displayed increased nuclear localization. Thus, by promoting cytosolic oligomerization, IP(6) binding is proposed to be a negative regulator of interactions of arrestin with plasma membrane and nuclear signaling proteins.

Animals↗
PubMed2006

Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex.

Adiponectin is a multifunctional adipokine that circulates as several oligomeric complexes in the blood stream. However, the molecular basis that regulates the production of the adiponectin oligomers remains largely elusive. We have shown previously that several conserved lysine residues (positions 68, 71, 80, and 104) within the collagenous domain of adiponectin are modified by hydroxylation and glycosylation (Wang, Y., Xu, A., Knight, C., Xu, L. Y., and Cooper, G. J. (2002) J. Biol. Chem. 277, 19521-19529). Here, we investigated the potential roles of these post-translational modifications in oligomeric complex formation of adiponectin. Gel filtration chromatography revealed that adiponectin produced from mammalian cells formed trimeric, hexameric, and high molecular weight (HMW) oligomeric complexes. These three oligomeric forms were differentially glycosylated, with the HMW oligomer having the highest carbohydrate content. Disruption of hydroxylation and glycosylation by substitution of the four conserved lysines with arginines selectively abrogated the intracellular assembly of the HMW oligomers in vitro as well as in vivo. In type 2 diabetic patients, both the ratios of HMW to total adiponectin and the degree of adiponectin glycosylation were significantly decreased compared with healthy controls. Functional studies of adiponectin-null mice revealed that abrogation of lysine hydroxylation/glycosylation markedly decreased the ability of adiponectin to stimulate phosphorylation of AMP-activated protein kinase in liver tissue. Chronic treatment of db/db diabetic mice with wild-type adiponectin alleviated hyperglycemia, hypertriglyceridemia, hepatic steatosis, and insulin resistance, whereas full-length adiponectin without proper post-translational modifications and HMW oligomers showed substantially decreased activities. Taken together, these data suggest that hydroxylation and glycosylation of the lysine residues within the collagenous domain of adiponectin are critically involved in regulating the formation of its HMW oligomeric complex and consequently contribute to the insulin-sensitizing activity of adiponectin in hepatocytes.

Adipocytes↗
PubMedDate not supplied

Regulation of connexin43 oligomerization is saturable.

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.

Cell Communication↗
PubMedDate not supplied

Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras.

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.

Animals↗
PubMed1992

Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.

Amino Acid Sequence↗
PubMed2002

Selective neuronal degeneration induced by soluble oligomeric amyloid beta protein.

The prevailing amyloid hypothesis for Alzheimer's disease (AD) holds that amyloid beta-protein (Abeta) causes neuronal degeneration by forming neurotoxic fibrillar structures. Yet, many aspects of AD pathology and symptoms are not well explained by this hypothesis. Here, we present evidence that neurotoxicity of soluble oligomeric Abeta closely corresponds to the selective neurodegeneration so distinctly manifest in AD. Selectivity was first observed in vitro, where only the human central nervous system neuronal cells were susceptible to soluble oligomeric Abeta. Furthermore, in mouse cerebral slice treated with soluble oligomeric Abeta, selective regiospecific toxicity was evident in the hippocampal CA1, a division important for memory, but not in the CA3 subfield. The fibrillar Abeta, however, killed neurons in all regions of the cerebral slice cultures and also in cerebellar slices. Remarkably, even at the highest soluble oligomeric Abeta concentrations, cerebellar neurons were completely spared, consistent with one of the hallmark features of AD pathology. Our observation of the selective neurodegeneration of soluble oligomeric Abeta to neurons involved in cognitive function may provide a new opportunity for the development of an effective AD therapy as well as elucidating the pathological mechanism of AD.

Alzheimer Disease↗
PubMed2004

Soluble oligomeric Abeta disrupts the protein kinase C signaling pathway.

Alzheimer's disease (AD) is characterized by selective neurodegeneration of neurons involved in cognitive function. Current hypothesis for AD etiology needs to be reconsidered because fibrillar Abeta cannot explain selective neurodegeneration. Recent evidence suggests oligomeric Abeta may be more relevant to AD etiology. Here we show signaling disruption induced by oligomeric Abeta. Using the MTT assay, NT2 showed greatest susceptibility to soluble oligomeric Abeta. In the kinase assay, treatment with either monomeric Abeta or fibrillar Abeta evoked no response in PKA, PKC and TK. Oligomeric Abeta treatment, however, inactivated membranous PKC but activated cytosolic PKC in NT2 within 24 h. Our data suggest that oligomeric Abeta may cause selective neurodegeneration through a PKC signaling, distinctive from fibrillar Abeta.

Amyloid beta-Peptides↗
PubMed1984

Oligomerization of simian virus 40 large T antigen is not necessarily repressed by temperature-sensitive A gene lesions.

Simian virus 40 large T antigen is a multifunctional protein which exists in different molecular weight forms. According to several reports, T antigen encoded by temperature-sensitive simian virus 40 A locus mutants (tsA) is unable to oligomerize into high-molecular-weight species. To try to correlate structural and functional properties, we selected tsA58 and tsA1499, both of which are heat sensitive for lytic growth, but only tsA58 is heat sensitive for transformation. Here we report that at permissive and nonpermissive temperatures, T antigen from tsA1499-infected monkey cells retained the ability to oligomerize, whereas reported previously, tsA58 T antigen failed to oligomerize at the nonpermissive temperature. Furthermore, we studied the formation of complexes between T antigen and the cellular p53 protein (T-p53) late in infection. Corresponding to its heat-stable oligomerization properties, T antigen encoded by tsA1499 formed T-p53 complexes regardless of temperature. In contrast, tsA58 encoded T-p53 complexes, preformed at the permissive temperature, remained heat stable after shifting up to the nonpermissive temperature; but at this temperature no new T-p53 complexes arose. The mutants did not replicate viral DNA at the nonpermissive temperature, suggesting that neither the oligomerization of T antigen nor the formation of T-p53 complexes seems to be sufficient for viral DNA replication or for the expression of late viral proteins.

Animals↗
PubMed1995

Determinants of human immunodeficiency virus type 1 envelope glycoprotein oligomeric structure.

Oligomerization of the human immunodeficiency virus type 1 envelope (env) glycoproteins is mediated by the ectodomain of the transmembrane glycoprotein gp41. We report that deletion of gp41 residues 550 to 561 resulted in gp41 sedimenting as a monomer in sucrose gradients, while the gp160 precursor sedimented as a mixture of monomers and oligomers. Deletion of the nearby residues 571 to 582 did not affect the oligomeric structure of gp41 or gp160, but deletion of both sequences resulted in monomeric gp41 and predominantly monomeric gp160. Deletion of residues 655 to 665, adjacent to the membrane-spanning sequence, partially dissociated the gp41 oligomer while not affecting the gp160 oligomeric structure. In contrast, deletion of residues 510 to 518 from the fusogenic hydrophobic N terminus of gp41 did not affect the env glycoprotein oligomeric structure. Even though the mutant gp160 and gp120 molecules were competent to bind CD4, the mutations impaired fusion function, gp41-gp120 association, and gp160 processing. Furthermore, deletion of residues 550 to 561 or 550 to 561 plus 571 to 582 modified the antigenic properties of the proximal residues 586 to 588 and the distal residues 634 to 664. Our results indicate that residues 550 to 561 are essential for maintaining the gp41 oligomeric structure but that this sequence and additional sequences contribute to the maintenance of gp160 oligomers. Residues 550 to 561 map to the N terminus of a putative amphipathic alpha-helix (residues 550 to 582), whereas residues 571 to 582 map to the C terminus of this sequence.

Amino Acid Sequence↗
PubMed1991

Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization.

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

Amino Acid Sequence↗
PubMed1994

p53 domains: structure, oligomerization, and transformation.

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

Animals↗
PubMed1999

Regulation of c-Fes tyrosine kinase and biological activities by N-terminal coiled-coil oligomerization domains.

The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains.

Animals↗
PubMed2001

Oligomerization of DH domain is essential for Dbl-induced transformation.

The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homology (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, and the DH domain, together with the immediately adjacent pleckstrin homology (PH) domain, constitutes the minimum module bearing transforming function. In the present study, we demonstrate that the onco-Dbl protein exists in oligomeric form in vitro and in cells. The oligomerization is mostly homophilic in nature and is mediated by the DH domain. Mutagenesis studies mapped the region involved in oligomerization to the conserved region 2 of the DH domain, which is located at the opposite side of the Rho GTPase interacting surface. Residue His556 of this region, in particular, is important for this activity, since the H556A mutant retained the GEF catalytic capability and the binding activity toward Cdc42 and RhoA in vitro but was deficient in oligomer formation. Consequently, the Rho GTPase activating potential of the H556A mutant was significantly reduced in cells. The focus-forming and anchorage-independent growth activities of onco-Dbl were completely abolished by the His556-to-Ala mutation, whereas the abilities to stimulate cell growth, activate Jun N-terminal kinase, and cause actin cytoskeletal changes were retained by the mutant. The ability of onco-Dbl to oligomerize allowed multiple Rho GTPases to be recruited to the same signaling complex, and such an ability is defective in the H556A mutant. Taken together, these results suggest that oligomerization of onco-Dbl through the DH domain is essential for cellular transformation by providing the means to generate a signaling complex that further augments and/or coordinates its Rho GTPase activating potential.

3T3 Cells↗
PubMed2002

Chk2 activation and phosphorylation-dependent oligomerization.

The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.

Animals↗
PubMed1992

Intramembrane helix-helix association in oligomerization and transmembrane signaling.

In spite of our greatly expanded knowledge of the primary structures of transbilayer receptor proteins, our knowledge of the tertiary and quaternary structures that define the biological activity of these receptors is scant. If we assume that the transmembrane regions of receptor proteins form stable alpha-helices regardless of the mechanism of insertion, a two-stage model of protein folding can be applied. For a multiple-helix protein, the two-stage model would predict that stable helical formation would be followed by an association of the helices to form the appropriate tertiary/quaternary structure. The two-stage model of protein folding is supported by various experiments with bacteriorhodopsin demonstrating that separate proteolytic fragments of bacteriorhodopsin can be refolded separately and can specifically recognize each other in order to associate and form a biologically active molecule. At the level of the bilayer, we propose that the energetics required for the association and packing of the helical transmembrane regions of a multiple-helix protein should not be significantly different from the association of separate single-helix proteins into an oligomer. Given the homogeneity in primary and secondary structure of the transmembrane regions of single-helix proteins, the association of multiple monomers may physically define high vs low affinity states and be a plausible mechanism of signal transduction. Increasing data suggest that oligomerization of receptor proteins may be involved in signal transduction. The transmembrane domains of receptor proteins appear to contain information critical to signaling and may be involved in a close contact site between receptors. This observation allows the two-stage model of protein folding for multiple-helix proteins to be directly applied to the oligomerization of single-helix receptor proteins. In addition, significant data suggest that the ectodomains and cytoplasmic domains are also involved in signaling and oligomerization of receptor molecules. In effect, the present data suggest that the most plausible model, both mechanistically and energetically, is one that includes both oligomerization and a global allosteric conformational change involving all of the defined domains of the receptor molecule. An oligomerization/conformational change model would predict that new sites of close contact would occur between the domains of the receptor molecule, some of which may be between the transmembrane helices. Therefore, experimenters should be able to generate peptides or small molecules that can specifically interfere with either the oligomerization or generation of new close-contact sites involved in the conformational change of the receptor that leads to signaling.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗