Membrane Retention Signal Research Articles

Cloning and identification of a novel cDNA coding thioredoxin-related transmembrane protein 2.

Thioredoxin plays an important role in various cellular processes through redox regulation. Here we report the molecular cloning and characterization of one member of the thioredoxin superfamily, designated as TMX2. The TMX2 cDNA consists of 1644 nucleotides and contains an open reading frame encoding a protein of 372 amino acids with a predicted molecular mass of 42.5 kDa and an isoelectric point of 8.94. The TMX2 protein may possess an N-terminal signal peptide, a potential transmembrane domain, an Myb DNA-binding domain repeat signature, a thioredoxin consensus pattern, an endoplasmic reticulum (ER) membrane retention signal (KKXX-like motif), and a dileucine motif in the tail. Northern blot analysis shows it is widely expressed in human tissues.

The Transmembrane Segment of Ryanodine Receptor Contains an Intracellular Membrane Retention Signal for Ca2+ Release Channel

The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.

Lipoprotein Sorting Signals Evaluated as the LolA-dependent Release of Lipoproteins from the Cytoplasmic Membrane of Escherichia coli

Escherichia coli lipoproteins are anchored to either the inner or outer membrane through fatty acyl chains covalently attached to an N-terminal cysteine. Aspartate at position 2 functions to retain lipoproteins in the inner membrane, although the retention is perturbed depending on the residue at position 3. We previously revealed that LolCDE and LolA play critical roles in this lipoprotein sorting. To clarify the sorting signals, the LolA-dependent release of lipoprotein derivatives having various residues at positions 2 and 3 was examined in spheroplasts. When the residue at position 3 was serine, only aspartate at position 2 caused the retention of lipoproteins in spheroplasts. We then examined the release of derivatives having aspartate at position 2 and various residues at position 3. Strong inner membrane retention occurred with a limited number of species of residues at position 3. These residues were present at position 3 of native lipoproteins having aspartate at position 2, whereas residues that inhibited the retention were not. It was also found that a strong inner membrane retention signal having residues other than aspartate at position 2 could be formed through the combination of the residues at positions 2 and 3. These results indicate that the inner membrane localization of native lipoproteins is ensured by the use of a limited number of strong inner membrane retention signals.

New functions for the NHERF family of proteins.

The sodium-hydrogen exchanger regulatory factor, NHERF (also called EBP50), was isolated initially as a necessary cofactor in cAMP-associated inhibition of the renal brush border sodium-hydrogen exchanger (NHE3) (1). Shortly thereafter, a second member of this family was cloned and characterized (NHERF2, also called E3KARP and TKA1) (2). NHERF and NHERF2 contain two tandem protein-protein interactive PDZ domains. Following the demonstration that these proteins bind ezrin, a new model was developed for regulation of NHE3 signaling by cAMP (3, 4), in which NHERF (or NHERF2), ezrin, and protein kinase A form a multiprotein signal complex linking NHE3 to the actin cytoskeleton. The formation of this complex is proposed to facilitate the phosphorylation and downregulation of NHE3. Recent studies have confirmed the basic elements of the model (4). This form of signal transduction, not previously considered in control of epithelial transporters, has generated considerable interest (5), and recent experiments have documented an association between NHERF and NHERF2 with a number of other transporters (4). While the precise relation between NHERF and these transport proteins remains to be determined, there are already preliminary data to suggest that NHERF interacts with these proteins in ways not directly linked to its role as an adaptor protein. A remarkable offshoot of the isolation of NHERF has been the discovery of its involvement with a broad array of biologic systems. At least three of the effects of NHERF are distinct from its regulation of renal transporters and deserve special comment. First, Katzenellenbogen and coworkers reported that estrogen stimulates the expression of NHERF in estrogen receptor-positive but not receptor-negative breast cancer cell lines and that antiestrogens can block this effect (6). Concurrent with these studies, we had isolated a large fragment of the mouse NHERF promoter and, given the above results, were surprised to find that it contains no recognizable estrogen response element and that estrogen fails to stimulate NHERF reporter constructs transiently expressed in estrogen receptor-positive cell lines (7). The mechanism of estrogen stimulation of NHERF remains to be determined; nonetheless, it is suggested that NHERF is involved in the signal-transduction pathway of estrogen. Recently, Stemmer-Rachamimov et al. reported a correlation between expression of NHERF and of estrogen receptors in breast carcinoma specimens (8). Others have made similar observations. The array of proteins stimulated by estrogen is finite, and identification of a new estrogen-responsive protein with a possible link to breast cancer is likely to stimulate research into the possible role of NHERF in breast, endometrium, and other estrogen-stimulated tissues. The relation between NHERF and the cystic fibrosis transmembrane regulator (CFTR) has been another area of increasing interest. CFTR functions as a cAMP-regulated chloride channel, and mutations in CFTR are causal in cystic fibrosis. CFTR contains a C-terminal NHERF consensus sequence and the two proteins bind with high affinity, an interaction that may help explain CFTR’s ability to regulate other transport proteins, including the epithelial sodium channel, the renal outer medullary potassium channel, and NHE3. Recent experiments have postulated two, not necessarily exclusive, roles for NHERF in CFTR function. Guggino, Stanton, and coworkers have proposed that NHERF functions as a membrane retention signal for CFTR (9). Foskett and coworkers suggest that NHERF facilitates the dimerization of CFTR and, as a consequence, full expression of chloride channel activity (10). As is the case for the relation between NHERF and estrogen signaling, the associations between NHERF and CFTR and their relevance to the complex physiologic derangements seen in cystic fibrosis are still unfolding.