Peptide probe study of the role of interaction between the cytoplasmic and transmembrane domains of the ryanodine receptor in the channel regulation mechanism

Ryanodine receptor (RyR) mutations linked with some congenital skeletal and cardiac diseases are localized to three easily definable regions: region 1 (N-terminal domain), region 2 (central domain), and a rather broad region 3 containing the channel pore. As shown in our recent studies, the interdomain interaction between regions 1 and 2 plays a critical role in channel regulation and pathogenesis. Here we present evidence that within region 3 there is a similar channel regulation mechanism mediated by an interdomain interaction. DP15, a peptide corresponding to RyR1 residues 4820-4841, produced significant activation of [3H]ryanodine binding above threshold Ca2+ concentrations (>or=0.3 microM), but MH mutations (L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identify the DP15 binding site(s) within RyR1, DP15 (labeled with a fluorescent probe Alexa Fluor 680 and a photoaffinity cross-linker APG) was cross-linked to RyR1, and the site of cross-linking was identified by gel analysis of fluorescently labeled proteolytic fragments with the aid of Western blotting with site-specific antibodies. The shortest fluorescently labeled band was a 96 kDa fragment which was stained with an antibody directed to the region of residues 4114-4142 of RyR1, indicating that the interaction between the region of residues 4820-4841 adjacent to the channel pore and the 96 kDa segment containing the region of residues 4114-4142 is involved in the mechanism of Ca2+-dependent channel regulation. In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channel activation similar to that of DP15.     

Hierarchy between the transmembrane and cytoplasmic domains in the regulation of syndecan-4 functions

Syndecan-4, a transmembrane heparan sulfate proteoglycan, plays a critical role in cell adhesion. Both the transmembrane and cytoplasmic domains of syndecan-4 are known to contribute to its functions, but the regulatory mechanisms underlying the functional interplay between the two domains were previously unclear. Here, we examined the functional relationship between these two domains. Fluorescence resonance energy transfer (FRET)-based assays showed that syndecan-4 expression enhanced RhoA activation. Furthermore, rat embryonic fibroblasts (REFs) plated on fibronectin fragments lacking the heparin-binding domain that interacts with syndecan-4 showed much lower RhoA activation than that in cells plated on full-length fibronectin, indicating that RhoA is involved in syndecan-4-mediated cell adhesion signaling. Syndecan-4 mutants defective in transmembrane domain-induced oligomerization and syndecan-4 phosphorylation-mimicking cytoplasmic domain mutants showed decreases in RhoA activation and RhoA-related functions, such as adhesion, spreading and focal adhesion formation, and subsequent increase in cell migration, but the inhibitory effect was much higher in cells expressing the transmembrane domain mutants. The cytoplasmic domain mutants (but not the transmembrane domain mutants) retained the capacity to form SDS-resistant dimers, and the cytoplasmic mutants showed less inhibition of syndecan-4-mediated protein kinase C activation compared to the transmembrane domain mutants. Finally, cytoplasmic domain activation failed to overcome the inhibition conferred by mutation of the transmembrane domain. Taken together, these data suggest that the transmembrane domain plays a major role in regulating syndecan-4 functions, and further show that a domain hierarchy exists in the regulation of syndecan-4.     

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.     

Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.     

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.     

Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits

In this study we have determined the position of the C-terminal end of the transmembrane domains of human integrin subunits (alpha2, alpha5, beta1, beta2) in microsomal membranes using the glycosylation mapping technique. In contrast to the common view, the transmembrane helices were found to extend roughly to Phe(1129) in alpha2, to Phe(1026) in alpha5, to Ile(757) in beta1, and to His(728) in beta2. The alpha-carbon of the conserved lysine present near the C-terminal end of the transmembrane helix (Lys(1125) in alpha2, Lys(1022) in alpha5, Lys(752) in beta1, and Lys(724) in beta2) is buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. A possible role for the conserved lysine in integrin function is discussed.     

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains

The proteins synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.     

pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119-144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced alpha-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.     

Interaction between transmembrane domains five and six of the alpha -factor receptor

The alpha-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae. Previous studies implicated the third intracellular loop of this receptor in G protein activation. Therefore, the roles of transmembrane domains five and six (TMD5 and -6) that bracket the third intracellular loop were analyzed by scanning mutagenesis in which each residue was substituted with cysteine. Out of 42 mutants examined, four constitutive mutants and two strong loss-of-function mutants were identified. Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes. Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C, L248C, or S251C mutations in TMD6. Also, the L226C mutation in TMD5 caused constitutive activity when combined with either the M250C or S251C mutations in TMD6. The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact. In support of this, cysteines substituted at position 223 in TMD5 and position 247 in TMD6 formed a disulfide bond, providing the first direct evidence of an interaction between these transmembrane domains in the alpha-factor receptor. Altogether, these results identify an important region of interaction between conserved hydrophobic regions at the base of TMD5 and TMD6 that is required for the proper regulation of receptor signaling.     

Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD.     

Evaluation of intramolecular interaction between complementary domains, connected with a flexible chain

A method for the evaluation of the effective binding parameters for the interaction between two complementary domains connected with a flexible chain is suggested. The calculations are based on the assumption that the chain is absolutely flexible and does not hinder free relative diffusion of the domains in the solution but does not allow the domains to move away from each other further than the length of the chain. Then, if the distance between the connected domains and the affinity of the interaction between disconnected domains are known, the suggested method allows calculation so-called "local concentration" of the domains relative to each other. On this basis, it is possible to estimate the upper limit to the fractional content of domains in complex, which, when constrained by the linking polypeptide chain, may be much higher than implied by the absolute concentrations of the domains.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.     

Replacement of the human insulin receptor transmembrane and cytoplasmic domains by corresponding domains of the oncogene product v-ros leads to accelerated internalization, degradation, and down-regulation

Internalization, degradation, and insulin-induced down-regulation of insulin receptors were studied comparatively in transformed Chinese hamster ovary (CHO) cell lines, CHO.T and CHO.IR.ros, respectively expressing either the wild-type human insulin receptor (hIR) or a mutated hybrid receptor in which the transmembrane and cytoplasmic domains of hIR were replaced by corresponding domains of the transforming protein p68gag-ros (v-ros) of avian sarcoma virus UR2. At 37 degrees C, degradation of insulin receptors photoaffinity labeled on the cell surface (440 kDa) was most rapid for the hybrid hIR.ros (t1/2 1.0 +/- 0.1 h), intermediate for the wild-type hIR (t1/2 2.7 +/- 0.5 h), and slowest for the endogenous CHO insulin receptors (t1/2 3.7 +/- 0.7 h). Initial intracellular accumulation of the hIR.ros hybrid was also most rapid, reaching maximal amounts in 20 min following which the receptors disappeared rapidly from the intracellular compartment. In contrast, intracellular accumulation of the receptors in the CHO.T and CHO cells was slower, reaching maximal amounts in 60 min, and rapid disappearance of the receptors from the intracellular compartment did not occur. Chloroquine, a lysosomotropic agent, inhibited degradation of both the wild-type hIR and the chimeric hIR.ros and increased their intracellular accumulation. However, the chloroquine effect was much more marked for the hIR.ros receptors whose intracellular accumulation was increased by greater than 300% (in comparison with approximately 60% increase for the wild-type hIR), demonstrating marked intracellular degradation of the hybrid hIR.ros at chloroquine-sensitive sites. Insulin-induced down-regulation of the cell surface hIR.ros (52% loss in 3 h) was also more marked than the wild-type hIR (approximately 30% loss in 3 h). Thus, in the hybrid hIR.ros receptor, which was shown previously to exhibit insulin-stimulated autophosphorylation and kinase activity but not insulin-stimulated metabolic function, the capacity for internalization and down-regulation is not only preserved but is also markedly accelerated. These findings suggest that 1) the postreceptor coupling mechanisms mediating insulin-induced receptor internalization, degradation, and down-regulation are different from those mediating metabolic functions; and 2) v-ros may contain the structural information directing accelerated receptor catabolism.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly ¹⁵N and ¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the ¹H, ¹³C, and ¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains

G-protein-coupled inwardly rectifying potassium channels (GIRK / Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K⁺ ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein βγ subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast gating. This result suggests that there is a cooperative movement of both of these domains to allow fast and efficient gating of GIRK channels.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Focus on composition and interaction potential of single-pass transmembrane domains

Transmembrane domains (TMD) connect the inner with the outer world of a living cell. Single TMD containing (bitopic) receptors are of particular interest, because their oligomerization seems to be a common activation mechanism in cell signaling. We analyzed the composition of TMDs in bitopic proteins within the proteomes of 12 model organisms. The average number of strongly polar and charged residues decreases during evolution, while the occurrence of a dimerization motif, GxxxG, remains unchanged. This may reflect the avoidance of unspecific binding within a growing receptor interaction network. In addition, we propose a new experimental approach for studying helix-helix interactions in giant plasma membrane vesicles using scanning fluorescence cross-correlation spectroscopy. Measuring eGFP/mRFP tagged versions of cytokine receptors confirms the homotypic interactions of the erythropoietin receptor in contrast to the Interleukin-4 receptor chains. As a proof of principle, by swapping the TMDs, the interaction potential of erythropoietin receptor was partially transferred to Interleukin-4 receptor α and vice versa. Non-interacting receptors can therefore serve as host molecules for TMDs whose oligomerization capability must be assessed. Computational analysis of the free energy gain resulting from TMD dimer formation strongly corroborates the experimental findings, potentially allowing in silico pre-screening of interacting pairs.     

Stable interactions between the transmembrane domains of the adenosine A2A receptor

G-protein-coupled receptors (GPCRs) must properly insert and fold in the membrane to adopt a stable native structure and become biologically active. The interactions between transmembrane (TM) helices are believed to play a major role in these processes. Previous studies in our group showed that specific interactions between TM helices occur, leading to an increase in helical content, especially in weakly helical TM domains, suggesting that helix–helix interactions in addition to helix–lipid interactions facilitate helix formation. They also demonstrated that TM peptides interact in a similar fashion in micelles and lipid vesicles, as they exhibit relatively similar thermal stability and α-helicity inserted in SDS micelles to that observed in liposomes. In this study, we perform an analysis of pairwise interactions between peptides corresponding to the seven TM domains of the human A_2A receptor (A_2AR). We used a combination of Förster resonance energy transfer (FRET) measurement and circular dichroism (CD) spectroscopy to detect and analyze these interactions in detergent micelles. We found that strong and specific interactions occur in only seven of the 28 possible peptide pairs. Furthermore, not all interactions, identified by FRET, lead to a change in helicity. Our results identify stabilizing contacts that are likely related to the stability of the receptor and that are consistent with what is known about the three-dimensional structure and stability of rhodopsin and the β₂ adrenergic receptor.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.