Filopodia formation driven by membrane glycoprotein M6a depends on the interaction of its transmembrane domains

Membrane glycoprotein M6a, which belongs to the tetraspan proteolipid protein family, promotes structural plasticity in neurons and cell lines by unknown mechanisms. This glycoprotein is encoded by Gpm6a, a stress‐regulated gene. The hippocampus of animals chronically stressed by either psychosocial or physical stressors shows decreased M6a expression. Stressed Gpm6a‐null mice develop a claustrophobia‐like phenotype. In humans, de novo duplication of GPM6A results in learning/behavioral abnormalities, and two single‐nucleotide polymorphisms (SNPs) in the non‐coding region are linked to mood disorders. Here, we studied M6a dimerization in neuronal membranes and its functional relevance. We showed that the self‐interaction of M6a transmembrane domains (TMDs) might be driving M6a dimerization, which is required to induce filopodia formation. Glycine mutants located in TMD2 and TMD4 of M6a affected its dimerization, thus preventing M6a‐induced filopodia formation in neurons. In silico analysis of three non‐synonymous SNPs located in the coding region of TMDs suggested that these mutations induce protein instability. Indeed, these SNPs prevented M6a from being functional in neurons, owing to decreased stability, dimerization or improper folding. Interestingly, SNP3 (W141R), which caused endoplasmic reticulum retention, is equivalent to that mutated in PLP1, W161L, which causes demyelinating Pelizaeus–Merzbacher disease.

     

The role of individual amino acids in the dimerization of CR4 and ACR4 transmembrane domains

CRINKLY4 is a growth factor-like plant receptor kinase designated as CR4 in Zea mays and ACR4 in Arabidopsis. Using the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment, we have previously demonstrated that the dimerization potential of the ACR4 transmembrane (TM) domain is significantly weaker than that of CR4 TM domain, even though 13 of the 24 residues are identical. Neither of the TM domains contain the GxxxG motif that has been shown to be important for the dimerization of the TM segments of several receptors. To further investigate the relationship between protein sequence and dimerization potential, we (a) mutated each of the 11 differing residues in the CR4 TM domain to the corresponding residue of ACR4 (b) made reciprocal mutations in ACR4 and (c) made hybrids consisting of half CR4 and half ACR4 TM domains. Our results suggest that most mutations in ACR4 or CR4 TM domains have low to moderate effects on the dimerization potential and that residues in the N-terminal half of the CR4 TM domain are important for dimerization.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

Investigation of Rhodobacter capsulatus PufX interactions in the core complex of the photosynthetic apparatus

The photosynthetic apparatus of purple bacteria in the genus Rhodobacter includes a core complex consisting of the reaction centre (RC), light-harvesting complex 1 (LH1), and the PufX protein. PufX modulates LH1 structure and facilitates photosynthetic quinone/quinol exchange. We deleted RC/LH1 genes in pufX ⁺ and pufX ⁺⁺ (merodiploid) strains of Rhodobacter capsulatus, which reduced PufX levels regardless of pufX gene copy number and location. Photosynthetic growth of RC-only strains and independent assembly kinetics of the RC and LH1 were unaffected by pufX merodiploidy, but the absorption spectra of strains expressing the RC plus either LH1 α or β indicated that PufX may influence bacteriochlorophyll binding environments. Significant self-association of the PufX transmembrane segment was detected in a hybrid protein expression system, consistent with a role of PufX in core complex dimerization, as proposed for other Rhodobacter species. Our results indicate that in R. capsulatus PufX has the potential to be a central, homodimeric core complex component, and its cellular level is increased by interactions with the RC and LH1.     

The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif

Experiments with the transmembrane (TM) domains of the glycoprotein (GP) Ib-IX complex have indicated that the associations between the TM domains of these subunits play an important role in the proper assembly of the complex. As a first step toward understanding these associations, we previously found that the Ibβ TM domain dimerized strongly in Escherichia coli cell membranes and led to Ibβ TM-CYTO (cytoplasmic domain) dimerization in the SDS-PAGE assay, while neither Ibα nor IX TM-CYTO was able to dimerize. In this study, we used the TOXCAT assay to probe the Ibβ TM domain dimerization interface by Ala- and Leu-scanning mutagenesis. Our results show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. Mutating either one of polar residues Gln129 or His139 to Leu or Ala disrupted Ibβ TM dimerization dramatically, indicating that polar residues might form part of the leucine zipper-based dimerization interface. Furthermore, these specific mutational effects in the TOXCAT assay were confirmed in the thiol-disulfide exchange and SDS-PAGE assays. The computational modeling studies further revealed that the most likely leucine zipper interface involves hydrogen bonding of Gln129 and electrostatic interaction of the His139 side chain. Correlation of computer modeling results with experimental mutagenesis studies on the Ibβ TM domain may provide insights for understanding the role of the association of TM domains on the assembly of GP Ib-IX complex.     

Dimerization properties of the transmembrane domains of Arabidopsis CRINKLY4 receptor-like kinase and homologs

CRINKLY4 (CR4) is a plant serine–threonine receptor kinase. In Zea mays, CR4 functions in the differentiation of the leaf epidermis and the aleurone cell layer and, in Arabidopsis thaliana, the ortholog ACR4 is involved in the development of the integument and seed coat. The Arabidopsis genome also encodes four CR4-related proteins (CRR) whose functions are not known. Based on studies of animal receptor kinase proteins it is likely that the molecular basis of function of CR4 and related proteins is mediated by receptor dimerization. The importance of the transmembrane (TM) domain in the dimerization of several receptor kinases has been demonstrated by the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment. In this study, we have used the TOXCAT assay to investigate the potential of the CR4 and CR4-related TM domains to homo-dimerize. Our investigation indicates that the CR4 TM domain and the CRR TM domains have higher propensities for homo-dimerization than the ACR4 TM domain. Interestingly, the dimerization potential of the ACR4 TM domain is significantly weaker even though 13 of 24 amino acids are identical to that of the CR4 TM domain. In order to determine the contributions of specific amino acids to the higher dimerization potential of CR4 compared to ACR4, mutations were made at specific sites in ACR4 TM domain and the strength of the dimer assessed by the TOXCAT assay. One mutation restored the activity to the CR4 level, while other mutations produced either no change or significantly increased the dimerization potential of the ACR4 TM domain. Our results indicate that the TM domains of CR4, ACR4 and the CRR receptor family of proteins have the intrinsic capacity to homo-dimerize, albeit with varying degrees of affinity.     

Strong oligomerization behavior of PDGFβ ?receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.     

A modified, dual reporter TOXCAT system for monitoring homodimerization of transmembrane segments of proteins

The TOXCAT assay system developed by Russ and Engelman [TOXCAT: a measure of transmembrane helix association in a biological membrane, Proc. Natl. Acad. Sci. USA 96 (1999) 863-868] provides an in vivo means of selecting for and evaluating the strength of interaction between identical transmembrane alpha-helices. In the course of utilizing TOXCAT to study the architecture of a sodium channel hNa(V)1.5, an apparently strong dimerization of two of its putative transmembrane segments was revealed. Following random mutagenesis of these regions, several amino acids critical for the observed dimerizations were identified. In order to develop a more efficient means of isolating mutations which specifically disrupt dimerization of these transmembrane segments without affecting their membrane-targeting properties, we developed a modification to the original TOXCAT design in which the C-terminal maltose binding protein moiety is replaced by the beta-lactamase. We show that this assay system is capable of simultaneously monitoring the integrity of the chimeric protein, its membrane insertion activity, and the ability of the transmembrane segment under study to dimerize.     

Motifs of serine and threonine can drive association of transmembrane helices

Known sequence motifs containing key glycine residues can drive the homo-oligomerization of transmembrane helices. To find other motifs, a randomized library of transmembrane interfaces was generated in which glycine was omitted. The TOXCAT system, which measures transmembrane helix association in the Escherichia coli inner membrane, was used to select high-affinity homo-oligomerizing sequences in this library. The two most frequently occurring motifs were SxxSSxxT and SxxxSSxxT. Isosteric mutations of any one of the serine and threonine residues to non-polar residues abolished oligomerization, indicating that the interaction between these positions is specific and requires an extended motif of serine and threonine hydroxyl groups. Computational modeling of these sequences produced several chemically plausible structures that contain multiple hydrogen bonds between the serine and threonine residues. While single serine or threonine side-chains do not appear to promote helix association, motifs can drive strong and specific association through a cooperative network of interhelical hydrogen bonds.     

The dimerization of PSGL‐1 is driven by the transmembrane domain via a leucine zipper motif

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is a homodimeric mucin ligand that is important to mediate the earliest adhesive event during an inflammatory response by rapidly forming and dissociating the selectin‐ligand adhesive bonds. Recent research indicates that the noncovalent associations between the PSGL‐1 transmembrane domains (TMDs) can substitute for the C320‐dependent covalent bond to mediate the dimerization of PSGL‐1. In this article, we combined TOXCAT assays and molecular dynamics (MD) simulations to probe the mechanism of PSGL‐1 dimerization. The results of TOXCAT assays and Martini coarse‐grained molecular dynamics (CG MD) simulations demonstrated that PSGL‐1 TMDs strongly dimerized in a natural membrane and a leucine zipper motif was responsible for the noncovalent dimerization of PSGL‐1 TMD since mutations of the residues that occupied a or d positions in an (abcdefg)n leucine heptad repeat motif significantly reduced the dimer activity. Furthermore, we studied the effects of the disulfide bond on the PSGL‐1 dimer using MD simulations. The disulfide bond was critical to form the leucine zipper structure, by which the disulfide bond further improved the stability of the PSGL‐1 dimer. These findings provide insights to understand the transmembrane association of PSGL‐1 that is an important structural basis for PSGL‐1 preferentially binding to P‐selectin to achieve its biochemical and biophysical functions.