Monte Carlo simulations of receptor dynamics: Insights into cell signaling

Many receptor-level processes involve the diffusion and reaction of receptors with other membrane-localized molecules. Monte Carlo simulation is a powerful technique that allows us to track the motions and discrete reactions of individual receptors, thus simulating receptor dynamics and the early events of signal transduction. In this paper, we discuss simulations of two receptor processes, receptor dimerization and G-protein activation. Our first set of simulations demonstrates how receptor dimerization can create clusters of receptors via partner switching and the relevance of this clustering for receptor cross-talk and integrin signaling. Our second set of simulations investigates the activation and desensitization of G-protein coupled receptors when either a single agonist or both an agonist and an antagonist are present. For G-protein coupled receptor systems in the presence of an agonist alone, the dissociation rate constant of agonist is predicted to affect the ratio of G-protein activation to receptor phosphorylation. Similarly, this ratio is affected by the antagonist dissociation rate constant when both agonist and antagonist are present. The relationship of simulation predictions to experimental findings and potential applications of our findings are also discussed.     

Adaptive coarse-grained Monte Carlo simulation of reaction and diffusion dynamics in heterogeneous plasma membranes

Background  

An adaptive coarse-grained (kinetic) Monte Carlo (ACGMC) simulation framework is applied to reaction and diffusion dynamics in inhomogeneous domains. The presented model is relevant to the diffusion and dimerization dynamics of epidermal growth factor receptor (EGFR) in the presence of plasma membrane heterogeneity and specifically receptor clustering. We perform simulations representing EGFR cluster dissipation in heterogeneous plasma membranes consisting of higher density clusters of receptors surrounded by low population areas using the ACGMC method. We further investigate the effect of key parameters on the cluster lifetime.     

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general.     

Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca²⁺ and K⁺ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163–202). The Ca²⁺-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and binding to negatively-charged groups on the membrane surface. The K⁺-induced reaction includes only dimerization and is shown to be due to screening interactions alone.The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca²⁺-induced reaction has a ‘bimolecular’ rate constant of 4.77 · 10⁸ M^?1 · s^?1 while the value for the K⁺-induced reaction is 7.05 · 10⁹ M^?1 · s^?1. These values are close to the limit of diffusion control (8.03 · 10⁹ M^?1 · s^?1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca²⁺-induced aggregation showed a break at 5°C. Above this temperature, the activation energy is low ($\text{+0.65}\text{kcal/mol}$), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing the energetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of ‘recognition sites’ on the membrane surface.     

Opposite Effects of the Melanocortin-2 (MC2) Receptor Accessory Protein MRAP on MC2 and MC5 Receptor Dimerization and Trafficking

MC2 (ACTH) receptors require MC2 receptor accessory protein (MRAP) to reach the cell surface. In this study, we show that MRAP has the opposite effect on the closely related MC5 receptor. In enzyme-linked immunosorbent assay and microscopy experiments, MC2 receptor was retained in the endoplasmic reticulum in the absence of MRAP and targeted to the plasma membrane with MRAP. MC5 receptor was at the plasma membrane in the absence of MRAP, but trapped intracellularly when expressed with MRAP. Using bimolecular fluorescence complementation, where one fragment of yellow fluorescent protein (YFP) was fused to receptors and another to MRAP, we showed that MC2 receptor-MRAP dimers were present at the plasma membrane, whereas MC5 receptor-MRAP dimers were intracellular. Both MC2 and MC5 receptors co-precipitated with MRAP. MRAP did not alter expression of β2-adrenergic receptors or co-precipitate with them. To determine if MRAP affects formation of receptor oligomers, we co-expressed MC2 receptors fused to YFP fragments in the presence or absence of MRAP. YFP fluorescence, reporting MC2 receptor homodimers, was readily detectable with or without MRAP. In contrast, MC5 receptor homodimers were visible in the absence of MRAP, but little fluorescence was observed by microscopic analysis when MRAP was co-expressed. Co-precipitation of differentially tagged receptors confirmed that MRAP blocks MC5 receptor dimerization. The regions of MRAP required for its effects on MC2 and MC5 receptors differed. These results establish that MRAP forms stable complexes with two different melanocortin receptors, facilitating surface expression of MC2 receptor but disrupting dimerization and surface localization of MC5 receptor.In mammals, the five members of the melanocortin (MC²) receptor family play diverse physiological roles. MC1 receptors (melanocyte-stimulating hormone receptors) control pigmentation in many animals, MC2 receptors (ACTH receptors) regulate adrenal corticosteroid synthesis, MC3 and MC4 receptors in brain influence food intake and energy expenditure, and MC5 receptors control exocrine gland secretion (1). Melanocortin receptors (MC1 through MC5) are structurally related G protein-coupled receptors that respond to agonists with an increase in cAMP. The receptors differ in their affinity for physiological agonists (α-, β-, and γ-melanocyte-stimulating hormone and ACTH) and antagonists (agouti and agouti-related protein).Unlike other melanocortin receptors, MC2 receptors are selectively regulated by ACTH. The MC2 receptor is also unusual in its requirement for an accessory protein, the MC2 receptor accessory protein (MRAP) (2). MRAP must be expressed with the MC2 receptor in order for the receptor to undergo glycosylation, traffic to the plasma membrane, bind ACTH, and stimulate adenylyl cyclase (2–4). Individuals with inactivating mutations of either the MC2 receptor or MRAP suffer from ACTH resistance and severe glucocorticoid deficiency (2).MRAP is a small protein with a conserved amino terminus, single membrane-spanning domain, and non-conserved carboxyl terminus that can also differ among splice variants. MRAP forms antiparallel homodimers, which are exceedingly rare structures, and it is these dimers that co-precipitate with the MC2 receptor (3). Mutagenic analysis has revealed that the carboxyl-terminal region of MRAP is not essential, but the amino-terminal and transmembrane regions are necessary for function (3–5). Deletion or alanine substitution of a critical four-amino acid segment, LDYI, at residues 18–21 of the mouse sequence, results in an MRAP molecule that facilitates expression of the MC2 receptor on the plasma membrane but does not allow the receptor to bind agonist or signal (4). MRAP is not required for cell surface expression of other melanocortin receptors and can inhibit expression or signaling in some cases (3, 6).The MC5 receptor was identified on the basis of its homology to other melanocortin receptors and is thought to be the ancestral ACTH (MC2) receptor (7). Analysis of MC5 receptor knock-out mice revealed that the MC5 receptor is important in controlling exocrine gland secretion (8) and behavioral responses depending on pheromones secreted by the preputial gland (9). MC2 and MC5 receptors are closely related, with 46% identity and 67% homology at the amino acid level, respectively. MC2 and MC5 receptors are both found on 3T3-L1 adipocytes and in some adipose tissues in animals (10, 11). Furthermore, MC2 and MC5 receptors are both expressed in adrenal cortex during embryonic development, when the MC5 receptor appears before the MC2 receptor (12). Mammalian MC2 and MC5 receptors are activated by different pro-opiomelanocortin peptides, responding physiologically to ACTH and melanocyte-stimulating hormone, respectively. Here we demonstrate that MC2 and MC5 receptors are also differentially regulated by MRAP, which exerts opposite effects on surface expression and dimerization of the two receptors.     

Activation of Neu (ErbB-2) Mediated by Disulfide Bond-Induced Dimerization Reveals a Receptor Tyrosine Kinase Dimer Interface

Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185^neu), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices.     

Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production

Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1^?/? and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1^?/? mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1^?/? mice. Furthermore, contrary to expectations, Trpc1^?/? BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1^?/? BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1^?/? mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.     

Theory and Simulations of Adhesion Receptor Dimerization on Membrane Surfaces

The equilibrium constants of trans and cis dimerization of membrane bound (2D) and freely moving (3D) adhesion receptors are expressed and compared using elementary statistical-thermodynamics. Both processes are mediated by the binding of extracellular subdomains whose range of motion in the 2D environment is reduced upon dimerization, defining a thin reaction shell where dimer formation and dissociation take place. We show that the ratio between the 2D and 3D equilibrium constants can be expressed as a product of individual factors describing, respectively, the spatial ranges of motions of the adhesive domains, and their rotational freedom within the reaction shell. The results predicted by the theory are compared to those obtained from a novel, to our knowledge, dynamical simulations methodology, whereby pairs of receptors perform realistic translational, internal, and rotational motions in 2D and 3D. We use cadherins as our model system. The theory and simulations explain how the strength of cis and trans interactions of adhesive receptors are affected both by their presence in the constrained intermembrane space and by the 2D environment of membrane surfaces. Our work provides fundamental insights as to the mechanism of lateral clustering of adhesion receptors after cell-cell contact and, more generally, to the formation of lateral microclusters of proteins on cell surfaces.     

C-tail mediated modulation of somatostatin receptor type-4 homo- and heterodimerizations and signaling

Somatostatin receptors show great diversity in response to agonist mediated receptor-specific homo- and heterodimerizations. Here, using photobleaching-fluorescence resonance energy transfer, immunocytochemistry, western blot and co-immunoprecipitation, we investigated dimerization, trafficking, coupling to adenylyl cyclase and signaling of human somatostatin receptor-4 (hSSTR4) in HEK-293 cells. We also determined the role of the C-tail of hSSTR4 on physiological responses of the cells. wt-hSSTR4 exogenously expressed in HEK-293 cells exhibits constitutive dimerization, inhibits forskolin-stimulated cAMP, and displays agonist dependent changes in pERK1/2 and pERK5 expressions. Upon C-tail deletion, the receptor loses membrane expression and ability to dimerize and inhibition of cAMP and pERK5 however, displays several-fold increases in the expression of pERK1/2. Chimeric hSSTR4 with the C-tail of hSSTR5 functions like wt-hSSTR4, in contrast, with the C-tail of hSSTR1 functions like C-tail deleted hSSTR4. hSSTR4 dimerization and signaling are associated with increased cyclin-dependent-kinase p27^kip1 expression and inhibition of the cell proliferation. We also report heterodimerization between hSSTR4/hSSTR5, but not between hSSTR4/hSSTR1, with significant changes in receptor functions. Taken together, these data define a novel mechanism for the role of hSSTR4 in cell proliferation and modulation of signaling pathways.     

Establishment of the ambient pH signaling complex in Aspergillus nidulans: PalI assists plasma membrane localization of PalH

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cys-containing motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both Saccharomyces cerevisiae and A. nidulans, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.     

Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G��s

Mahogunin ring finger-1 (MGRN1) is a RING domain-containing ubiquitin ligase mutated in mahoganoid, a mouse mutation causing coat color darkening, congenital heart defects, high embryonic lethality, and spongiform neurodegeneration. The melanocortin hormones regulate pigmentation, cortisol production, food intake, and body weight by signaling through five G protein-coupled receptors positively coupled to the cAMP pathway (MC1R–MC5R). Genetic analysis has shown that mouse Mgrn1 is an accessory protein for melanocortin signaling that may inhibit MC1R and MC4R by unknown mechanisms. These melanocortin receptors (MCRs) regulate pigmentation and body weight, respectively. We show that human melanoma cells express 4 MGRN1 isoforms differing in the C-terminal exon 17 and in usage of exon 12. This exon contains nuclear localization signals. MGRN1 isoforms decreased MC1R and MC4R signaling to cAMP, without effect on β₂-adrenergic receptor. Inhibition was independent on receptor plasma membrane expression, ubiquitylation, internalization, or stability and occurred upstream of Gα_s binding to/activation of adenylyl cyclase. MGRN1 co-immunoprecipitated with MCRs, suggesting a physical interaction of the proteins. Significantly, overexpression of Gα_s abolished the inhibitory effect of MGRN1 and decreased co-immunoprecipitation with MCRs, suggesting competition between MGRN1 and Gα_s for binding to MCRs. Although all MGRN1s were located in the cytosol in the absence of MCRs, exon 12-containing isoforms accumulated in the nuclei upon co-expression with the receptors. Therefore, MGRN1 inhibits MCR signaling by a new mechanism involving displacement of Gα_s, thus accounting for key features of the mahoganoid phenotype. Moreover, MGRN1 might provide a novel pathway for melanocortin signaling from the cell surface to the nucleus.     

An Essential Role for the K+-dependent Na+/Ca2+-exchanger,NCKX4, in Melanocortin-4-receptor-dependent Satiety

K⁺-dependent Na⁺/Ca²⁺-exchangers are broadly expressed in various tissues, and particularly enriched in neurons of the brain. The distinct physiological roles for the different members of this Ca²⁺ transporter family are, however, not well described. Here we show that gene-targeted mice lacking the K⁺-dependent Na⁺/Ca²⁺-exchanger, NCKX4 (gene slc24a4 or Nckx4), display a remarkable anorexia with severe hypophagia and weight loss. Feeding and satiety are coordinated centrally by melanocortin-4 receptors (MC4R) in neurons of the hypothalamic paraventricular nucleus (PVN). The hypophagic response of Nckx4 knock-out mice is accompanied by hyperactivation of neurons in the PVN, evidenced by high levels of c-Fos expression. The activation of PVN neurons in both fasted Nckx4 knock-out and glucose-injected wild-type animals is blocked by Ca²⁺ removal and MC4R antagonists. In cultured hypothalamic neurons, melanocyte stimulating hormone induces an MC4R-dependent and sustained Ca²⁺ signal, which requires phospholipase C activity and plasma membrane Ca²⁺ entry. The Ca²⁺ signal is enhanced in hypothalamic neurons from Nckx4 knock-out animals, and is depressed in cells in which NCKX4 is overexpressed. Finally, MC4R-dependent oxytocin expression in the PVN, a key essential step in satiety, is prevented by blocking phospholipase C activation or Ca²⁺ entry. These findings highlight an essential, and to our knowledge previously unknown, role for Ca²⁺ signaling in the MC4R pathway that leads to satiety, and a novel non-redundant role for NCKX4-mediated Ca²⁺ extrusion in controlling MC4R signaling and feeding behavior. Together, these findings highlight a novel pathway that potentially could be exploited to develop much needed new therapeutics to tackle eating disorders and obesity.     

N-Ras Forms Dimers at POPC Membranes

Ras is a central regulator of cellular signaling pathways. It is mutated in 20–30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between β2 and β3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.     

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange,a Molecular Dynamics Study

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys⁵⁶⁷–Cys⁵⁸¹ bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.     

HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Nef, a human immunodeficiency virus type 1 (HIV-1) accessory factor capable of interaction with a diverse array of host cell signaling molecules, is essential for high-titer HIV replication and AIDS progression. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher-order oligomers in HIV-infected cells, which may be required for both immune and viral receptor downregulation as well as viral replication. Using bimolecular fluorescence complementation, we provide the first direct evidence for Nef dimers within HIV host cells and identify the structural requirements for dimerization in vivo. Bimolecular fluorescence complementation analysis shows that the multiple hydrophobic and electrostatic interactions found within the dimerization interface of the Nef X-ray crystal structure are essential for dimerization in cells. Nef dimers localized to the plasma membrane as well as the trans-Golgi network, two subcellular localizations essential for Nef function. Mutations in the Nef dimerization interface dramatically reduced both Nef-induced CD4 downregulation and HIV replication. Viruses expressing dimerization-defective Nef mutants were disabled to the same extent as HIV that fails to express Nef in terms of replication. These results identify the Nef dimerization region as a potential molecular target for antiretroviral drug discovery.     

Monte Carlo simulations of plasma membrane corral-induced EGFR clustering

Experimental evidence suggests that the cell membrane is a highly organized structure that is compartmentalized by the underlying membrane cytoskeleton (MSK). The interaction between the cell membrane and the cytoskeleton led to the “picket-fence” model, which was proposed to explain certain aspects of membrane compartmentalization. This model assumes that the MSK hinders and confines the motion of receptors and lipids to compartments in the membrane. However, the impact of the MSK on receptor clustering, aggregation, and downstream signaling remains unclear. For example, some evidence suggests that the MSK enhances dimerization, while other evidence suggests decreased dimerization and signaling. Herein, we use computational Monte Carlo simulations to examine the effects of MSK density and receptor concentration on receptor dimerization and clustering. Preliminary results suggest that the MSK may have the potential to induce receptor clustering, which is a function of both picket-fence density and receptor concentration.     

Mechanism of dimerization of the human melanocortin 1 receptor

The melanocortin 1 receptor (MC1R) is a dimeric G protein-coupled receptor expressed in melanocytes, where it regulates the amount and type of melanins produced and determines the tanning response to ultraviolet radiation. We have studied the mechanisms of MC1R dimerization. Normal dimerization of a deleted mutant lacking the seventh transmembrane fragment and the C-terminal cytosolic extension excluded coiled-coil interactions as the basis of dimerization. Conversely, the electrophoretic pattern of wild type receptor and several Cys → Ala mutants showed that four disulfide bonds are established between the monomers. Disruption of any of these bonds abolished MC1R function, but only the one involving Cys35 was essential for traffic to the plasma membrane. A quadruple Cys35-267-273-275Ala mutant migrating as a monomer in SDS-PAGE in the absence of reducing agents was able to dimerize with WT, suggesting that in addition to disulfide bond formation, dimerization involves non-covalent interactions, likely of domain swap type.     

Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn²¹¹ residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved ²¹¹NDS N-glycosylation motif, but not other N-glycosylation sites (Asn²⁶⁰, Asn³⁷¹, and Asn³⁹⁴), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr⁴⁸⁴, Tyr⁵²⁰, Tyr⁷⁹², and Tyr⁷⁹⁷. The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved ²¹¹NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.     

A comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes

Plasma membrane-associated ATPase obtained from cauliflower (Brassica oleraceae L.) florets isolated and assayed by several different procedures was stimulated 150 to 400% by K⁺. In contrast, winter wheat (Triticum aestivum L. cv. Kharkov 22 MC) shoot and root ATPase obtained by the same methods exhibited only 10 to 25% stimulation by K⁺. The level of K⁺-stimulation of the wheat enzyme was not significantly increased by purifying the crude microsomal membrane fraction using sucrose density gradients. ATPase associated with density gradient-purified cauliflower membranes was inhibited by Ca²⁺, high ATP concentration in the presence of low Mg²⁺, and by several metabolic inhibitors. In contrast, the wheat enzyme was largely unaffected by all of these treatments. The plasma membranes of intact wheat and cauliflower cells gave a positive reaction with the plasma membrane-specific, phosphotungstic acid-chromic acid stain (PACP). A high proportion of the cauliflower membrane vesicles in the putative plasma membrane-enriched fraction stained with PACP, whereas only a small proportion of the wheat membrane vesicles reacted positively with PACP. These results indicate that a plasma membrane-enriched fraction has been isolated successfully from cauliflower floret tissue, but that none of the procedures used effectively separate plasma membranes from homogenates of wheat shoots and roots.