Transmembrane Superfamily Research Articles

TM9SF1 promotes bladder cancer cell growth and infiltration.

Bladder cancer (BC) is the most common urological tumor. It has a high recurrence rate, displays tutor heterogeneity, and resists chemotherapy. Furthermore, the long-term survival rate of BC patients has remained unchanged for decades, which seriously affects the quality of patient survival. To improve the survival rate and prognosis of BC patients, it is necessary to explore the molecular mechanisms of BC development and progression and identify targets for treatment and intervention. Transmembrane 9 superfamily member 1 (TM9SF1), also known as MP70 and HMP70, is a member of a family of nine transmembrane superfamily proteins, which was first identified in 1997. TM9SF1 can be expressed in BC, but its biological function and mechanism in BC are not clear. To investigate the biological function and mechanism of TM9SF1 in BC. Cells at 60%-80% confluence were transfected with lentiviral vectors for 48-72 h to achieve stable TM9SF1 overexpression or silencing in three BC cell lines (5637, T24, and UM-UC-3). The effect of TM9SF1 on the biological behavior of BC cells was then investigated through CCK8, wound-healing assay, transwell assay, and flow cytometry. Overexpression of TM9SF1 increased the in vitro proliferation, migration, and invasion of BC cells by promoting the entry of BC cells into the G2/M phase. Silencing of TM9SF1 inhibited in vitro proliferation, migration, and invasion of BC cells and blocked BC cells in the G1 phase. TM9SF1 may be an oncogene in BC.

α1 Proline 277 Residues Regulate GABAAR Gating through M2-M3 Loop Interaction in the Interface Region.

Cys-loop receptors are a superfamily of transmembrane, pentameric receptors that play a crucial role in mammalian CNS signaling. Physiological activation of these receptors is typically initiated by neurotransmitter binding to the orthosteric binding site, located at the extracellular domain (ECD), which leads to the opening of the channel pore (gate) at the transmembrane domain (TMD). Whereas considerable knowledge on molecular mechanisms of Cys-loop receptor activation was gathered for the acetylcholine receptor, little is known with this respect about the GABAA receptor (GABAAR), which mediates cellular inhibition. Importantly, several static structures of GABAAR were recently described, paving the way to more in-depth molecular functional studies. Moreover, it has been pointed out that the TMD-ECD interface region plays a crucial role in transduction of conformational changes from the ligand binding site to the channel gate. One of the interface structures implicated in this transduction process is the M2-M3 loop with a highly conserved proline (P277) residue. To address this issue specifically for α1β2γ2L GABAAR, we choose to substitute proline α1P277 with amino acids with different physicochemical features such as electrostatic charge or their ability to change the loop flexibility. To address the functional impact of these mutations, we performed macroscopic and single-channel patch-clamp analyses together with modeling. Our findings revealed that mutation of α1P277 weakly affected agonist binding but was critical for all transitions of GABAAR gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α1P277 at the interface is strongly involved in regulating the receptor gating.

An investigation into the allosteric mechanism of GPCR A2A adenosine receptor with trajectory-based information theory and complex network model

G protein-coupled receptors (GPCRs), a large superfamily of transmembrane (TM) proteins, allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to effector proteins in the intracellular (IC) domain, therefore forming the largest class of drug targets. The A2A adenosine receptor (A2AAR), a class-A GPCR, has been extensively studied as it offers numerous possibilities for therapeutic applications. However, the mechanism of allosteric communication between EC and IC domains is not completely clear. In this work, we utilize torsional mutual information to quantify the correlated motions of residue pairs from its molecular dynamics (MD) simulation trajectories, and further use the complex network model to obtain allosteric pipelines and hubs. The identified allosteric communication pipelines mainly transmit the signal from EC domain to the cytoplasmic ends of TM helix 5 (TM5), TM6 and TM7. The allosteric hubs, mostly located at TM5, TM6 and TM7, play an important role in mediating allosteric signal transmission to keep the receptor rigid and prevent G protein from binding to IC domain, which can explain the reason why their mutations distant from ligand-binding site do not affect the ligand binding affinity but affect the ligand efficacy. Additionally, we identify the key residues located in antagonist ZM241385 binding pocket which mediate multiple allosteric pathways and have been experimentally proven to play a critical role in affecting the ligand potency. This study is helpful for understanding the allosteric communication mechanism of A2AAR, and can provide valuable information for the structure-based drug design of GPCRs. Communicated by Ramaswamy H. Sarma

KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

BackgroundKAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.MethodsWe used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.ResultsWe demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.ConclusionsThese novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.

Targeting Proteases in Atherosclerosis

The initiation, progression, and complications of atherosclerosis are mediated by a complex interplay of hematopoietic cells, vascular cells, and the extracellular matrix in response to metabolic (eg, hyperlipidemia, diabetes mellitus) and environmental (eg, smoking) injury.1 Antiatherosclerotic therapies are currently limited to statins and their lipid-lowering and pleiotropic anti-inflammatory effects, to angiotensin-converting enzyme inhibitors and their local effects on vascular cell proliferation and plaque progression independently of blood pressure lowering, and to antiplatelet agents and their role in preventing the thrombotic complications of plaque rupture and superficial endothelial cell (EC) erosion.2,3 However, although these therapies have been shown to reduce event rates by ≈20% to 40% in randomized, placebo-controlled clinical trials, absolute event rates remain high. For example, recurrent ischemic events after index acute coronary syndrome presentation occur in >20% of patients by 36 months despite optimal medical therapy and revascularization.4 This failure of drug and device therapies represents an enormous unmet clinical need. In contrast to effective disease-modifying therapies in psoriasis, rheumatoid arthritis, and multiple sclerosis, targeted antiatherosclerotic therapies have remained elusive. In this issue of Circulation , Miyazaki and colleagues5 have identified m-calpain (calpain 2 and its regulatory domain, calpain 4) as a novel protease target for the modulation of atherogenesis. Article see p 2522 After 7 transmembrane superfamily receptors (G protein–coupled receptors), proteases are one of the most attractive pharmacological targets.6 The US market for protease-targeted drugs in 2009 was approximately $11 billion, with the major …

A Conserved Transmembrane Domain Interface Regulates Integrin Function

Integrins are a superfamily of transmembrane (TM) α/β heterodimers that mediate fundamental cellular adhesive functions. Platelet integrins, for example, mediate stable platelet adhesion to collagen and fibronectin and the formation of stable platelet aggregates. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. An essential feature of this equilibrium is interaction of the integrin α and β subunit TM domains. Thus, when integrins are inactive, the α and β TM domains are in proximity, but they separate when integrins assume an active conformation. Moreover, inducing TM domain separation alone is sufficient to cause integrin activation. Previously, we reported that the TM domains of the platelet integrin αIIbβ3 interact both heteromerically and homomerically and that the strength of their heteromeric interaction is necessarily weak to allow regulated TM domain separation. To address whether these observations can be extended to the other members of the integrin superfamily, we focused initially on αvβ3, α2β1 and α5β1, integrins present in platelets, using a dominant-negative ToxR-based assay. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it dimerizes in the inner membrane of E. coli. By co-expressing wild-type ToxR with either wild-type ToxR or an R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure the homomeric and heteromeric interaction of each integrin TM domain. Using alanine and leucine scanning mutagenesis, we found that like αIIb, homo-oligomerization of other integrin α subunit TM domains is preferred over hetero-oligomerization, and that the relative strength of homo-oligomerization correlates with the presence of a canonical small residue-xxx-small residue motif followed one turn of the TM helix by a leucine (G, A, S-xxx-G-xxx-L). This motif also mediates the hetero-oligomerization of these TM domains with either β3 or β1. By contrast, a different motif (V-xxx-I-xxx-G) mediates the heteromeric interaction of both β3 and β1 with their complementary α subunits. Mutations that disrupt either the αIIb or β3 interaction motif induce constitutive αIIbβ3 activation. To determine if this is also the case for β1-containing integrins, we introduced disruptive interfacial mutations into the full-length integrins and expressed the mutants in either the β1-deficient Jurkat A1 cells or in HEK293 suspension cells. We found that the β1 mutations V716A, I720A and G724L caused a substantial increase in the static adhesion of A1 cells to laminin, fibronectin, the α4β1-specific peptide H1, as well as type I, II and type IV collagen, whereas mutation of the canonical G-xxx-G motif did not. On the other hand, an increase in binding to type I collagen and fibronectin was observed for mutations of the interfacial α2 residues S1009, G1013, and L1017 and the interfacial α5 residues A964, G968, and L972, respectively. Thus, our studies indicate that β1 and β3 integrins employ a novel, specific, and conserved reciprocating ‘large-small’ TM packing interface that interacts less strongly than the canonical small-residue-xxx-small residue motif. It is also noteworthy that this interface is present in all integrins except β4 and is overrepresented in databases of TM helix-helix interaction as well. Accordingly, it is likely that this type of interface evolved to mediate TM domain interactions that are capable of regulation.

Epithelial membrane protein-2 is expressed in discrete anatomical regions of the eye

Epithelial membrane protein-2 (EMP2) is a member of the four transmembrane superfamily (TM4SF) and is thought to mediate trafficking of diverse proteins such as α6β1 integrin and MHC class I to lipid raft microdomains. EMP2 has also recently been recognized as a putative tumor suppressor gene in certain model systems. Normally, EMP2 is expressed at discrete locations in the body including high levels in the eye, lung, heart, thyroid, and uterus. Here we examine in detail the subanatomic distribution of EMP2 in murine and human ocular tissue. We observe that EMP2 is localized to epithelial layers of the cornea, ciliary body, and retinal pigmented epithelium–choroid, the stromal layers of the sclera, and the nerve fiber layer of the retina and optic nerve. This distribution is distinct from other TM4SF proteins and may relate to a role in apical membrane recycling.

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor.

Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Structural models of the transmembrane glycoproteins (GP-2) of the Arenaviruses, lymphochoriomeningitis virus (LCMV) and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

Observations of light-induced structural changes of retinal within rhodopsin.

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

PATHOGENESIS OF ADRENOCORTICAL INCIDENTALOMAS AND GENETIC SYNDROMES ASSOCIATED WITH ADRENOCORTICAL NEOPLASMS

The study of genetic syndromes associated with adrenocortical tumors (Beckwith-Wiedemann, Li-Fraumeni, McCune-Albright, Carney, and multiple endocrine neoplasia type 1) has shed light on the molecular basis of tumorigenesis. Abnormalities at the 11p15 locus appear as crucial and frequent events found specifically in malignant, sporadic tumors, leading to overexpression of a growth-promoting factor and loss of expression of tumor suppressor genes. In benign tumors, the cAMP pathway can be exacerbated in an ACTH-independent manner when various membrane receptors of the seven transmembrane superfamily are "illegitimately" expressed.

The amyloid precursor protein of Alzheimer's disease and the Abeta peptide.

Alzheimer's disease is characterized by the accumulation of beta amyloid peptides in plaques and vessel walls and by the intraneuronal accumulation of paired helical filaments composed of hyperphosphorylated tau. In this review, we concentrate on the biology of amyloid precursor protein, and on the central role of amyloid in the pathogenesis of Alzheimer's disease. Amyloid precursor protein (APP) is part of a super-family of transmembrane and secreted proteins. It appears to have a number of roles, including regulation of haemostasis and mediation of neuroprotection. APP also has potentially important metal and heparin-binding properties, and the current challenge is to synthesize all these varied activities into a coherent view of its function. Cleavage of amyloid precursor protein by beta-and gamma-secretases results in the generation of the Abeta (betaA4) peptide, whereas alpha-secretase cleaves within the Abeta sequence and prevents formation from APP. Recent findings indicate that the site of gamma-secretase cleavage is critical to the development of amyloid deposits; Abeta1-42 is much more amyloidogenic than Abeta1-40. Abeta1-42 formation is favoured by mutations in the two presenilin genes (PS1 and PS2), and by the commonest amyloid precursor protein mutations. Transgenic mouse models of Alzheimer's disease incorporating various mutations in the presenilin gene now exist, and have shown amyloid accumulation and cognitive impairment. Neurofibrillary tangles have not been reproduced in these models, however. While aggregated Abeta is neurotoxic, perhaps via an oxidative mechanism, the relationship between such toxicity and neurofibrillary tangle formation remains a subject of ongoing research.