Conserved Membrane Proximal Region Research Articles

The Membrane Proximal Region of the Cannabinoid Receptor CB1 N-Terminus Can Allosterically Modulate Ligand Affinity

The human cannabinoid receptor, CB1, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB1 N-terminus in modulating ligand binding to the receptor. Although most of the CB1 N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB1 receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB1, and this C98-C107 disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C98-C107 disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C98-C107 disulfide also alters the effects of allosteric ligands for CB1, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB1 and suggest that the CB1 N-terminal MPR may be an area through which allosteric modulators can act.

Disk large protein 1 promotes the initiation of isotype-switched IgG-BCR signaling (50.15)

Abstract B cell memory is critical for the protection from many infectious diseases and is the basis of many current vaccines. Memory B cells expressing high-affinity, IgG-BCRs give rise to the rapid production of high-affinity IgG antibody recall responses, the mechanisms by which the IgG BCRs function to enhance B cell responses are not understood. Studies in mouse models showed that the cytoplasmic tail of membrane-bound IgG (mIgG) is both required and sufficient for the enhanced IgG antibody recall response. Recently, we showed that the conserved membrane proximal region of the mIgG1 cytoplasmic tail, a region not previously appreciated to play a role in BCR signaling, was both necessary and sufficient to dramatically enhance the early events in the initiation of B cell activation. We now identify Dlg1 as a specific, antigen-induced binding partner of the mIgG1 cytoplasmic tail. We demonstrate that Dlg1 and mIg associate through a mechanism involving the binding of the N-terminal and PDZ domains of Dlg1 to the membrane proximal region of the mIgG1 tail. In response to antigens, Dlg1 is rapidly recruited to IgG1-BCR microcluster and then the IgG1-B cell immune synapse. Using live B cell imaging by fluorescence lifetime imaging microscopy we observed that antigen binding induces a molecular complex of Dlg1 and IgG-BCR. Our further evidence suggests that Dlg1 serves as a BCR adaptor stabilizing the formation of IgG-BCR oligomers and facilitating the assembly of signalosome.

Both lipid environment and pH are critical for determining physiological solution structure of 3‐D‐conserved epitopes of the HIV‐1 gp41‐MPER peptide P1

In terms of background, the solution structure of monomeric peptide P1 (residues 649-683), located in the conserved membrane proximal region (MPER) of HIV-1 envelope glycoprotein gp41, is first reported here in dodecylphosphocholine (DPC) micelles. P1 is the minimal MPER region that permits interaction with the mucosal galactosyl ceramide HIV-receptor; it also contains epitopes recognized by major gp41-specific, broadly neutralizing immunoglobulin Gs (IgGs), 2F5 and 4E10, determinant in HIV fusion/infection. Our principal findings were as follows: the structural stability of P1 is pH dependent, as the alpha-helix comprising Q653 I682, present at pH 3.3, is destabilized at higher pH values. At pH 6, the E-rich N-terminal half of P1 (residues 650-666), partially overlapping the 2F5-specific epitope, becomes fully disordered, while the W-rich C-terminal half conserves two shorter helices (W666-W670 and W672-W680), separated by a well-defined bend overlapped by the 4E10-specific epitope. The two IgGs bind to P1 on DPC micelles with binding parameters (K(d)) in the nanomolar range. Next, P1 was derivatized with phosphatidylethanolamine at its C terminal and inserted into liposomes of varied lipid composition, thereby enabling P1 to move laterally. Alternatively, an infectious virus-binding assay was established. The K(d) of both 2F5 and 4E10 IgGs measured on viral liposome and virus are similar and much lower than for the binding of the free peptide. In conclusion, P1, in a lipid environment, is an optimized MPER-derived peptide suitable for designing an immunogen inducing broadly neutralizing antibodies to HIV.

De Novo Expression of the Integrin α5β1 Regulates αvβ3-mediated Adhesion and Migration on Fibrinogen

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.

Human GM-CSF induces HIV-1 LTR by multiple signalling pathways

Human immunodeficiency virus type-1 (HIV-1) gene expression is known to be affected by numerous cytokines or growth factors. However, the effect of granulocyte–macrophage colony-stimulating factor (GM-CSF) on long terminal repeat (LTR)-mediated transcription of HIV-1 still remains unknown. By transient transfection experiments with HIV-1 LTR reporter constructs, we showed that strong LTR-mediated activation was induced by GM-CSF in mouse Ba/F3 cells expressing human GM-CSF receptors (GM-CSFR). Mutational analysis of the HIV-1 LTR reporters revealed that both NF-κB and Sp1 binding sites play important roles as positive regulatory elements. Analysis of various mutants of the cytoplasmic region of GM-CSFR indicated that both the conserved membrane proximal region and tyrosine residues located in the distal part of the β subunit were required for HIV-1 LTR activation. Possible involvement of MAPK and PI3-K signalling pathways was suggested by the partial inhibition by wortmannin, a specific inhibitor of the PI3-K pathway, and enhancement by constitutively active MEK1, of HIV-1 LTR activation. However, the MEK1 pathway is not essential since MEK1 inhibitor PD98059 did not suppress GM-CSF-induced HIV-1-LTR activation. Further analyses of GM-CSFR mutants suggested that some other unknown signalling pathway also participates in GM-CSF-induced HIV-1 LTR activation. Taken together, the data suggest that GM-CSF could upregulate the LTR-driven transcription of HIV-1 through modulation of NF-κB and SP1 by multiple signalling pathways.

Identification of novel interaction partners for the conserved membrane proximal region of α-integrin cytoplasmic domains

The α3Aβ1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like α2β1, α5β1 and α6Aβ1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the α3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of α subunits. The conserved membrane proximal region of the α3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.