Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.     

Analysis of the tumor-associated antigen TSP-180. Identity with alpha 6-beta 4 in the integrin superfamily

The tumor-associated antigen complex, TSP-180, was previously defined in carcinoma cell lines and found to be expressed in higher amounts in tumor than in normal tissue. Here, the mouse TSP-180 complex is shown to consist of three related proteins (bands 1, 2, and 3) associated with a distinct protein (band 5) that is probably derived from a precursor protein (band 4). All of these proteins are cell surface glycoproteins, and the largest protein (band 1) can be readily labeled with 32PO4. The mouse TSP-180 complex described here strongly resembles the recently described human integrin alpha 6-beta 4 complex. This homology was confirmed using two distinct rat anti-alpha 6 monoclonal antibodies, each of which recognized both human alpha 6-beta 4 and mouse TSP-180 complexes. Furthermore, the TSP-180 band 5 protein (mouse alpha 6) had an N-terminal sequence identical to that of human alpha 6. Finally, two different monoclonal antibodies are described, 346-11A and 439-9B, which directly recognize the multiple forms of mouse and human beta 4 proteins, respectively.     

Agonist- and competitive antagonist-induced movement of loop 5 on the alpha subunit of the neuronal alpha4beta4 nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that rapidly convert a chemical signal into an electrical signal. Although the structure of the nAChR is quite well described, the coupling between agonist binding and channel gating is still under debate. In this study, we probed local conformational transitions on the neuronal α4β4 nAChR by specifically tethering a conformation-sensitive fluorescent dye on αG98C located on loop 5 (L5), and simultaneously monitoring fluorescence intensity and current after expression in Xenopus oocytes. The potency of acetylcholine (ACh) was significantly higher in the cysteine mutant and further increased upon tetramethylrhodamine-6-maleimide labeling, suggesting a role of L5 in binding or gating. Structural reorganizations of L5 were shown to occur upon activation, as revealed by the fluorescence intensity increase during ACh exposure. Fluorescence changes were also detected at ACh concentrations lower than needed for current activation, suggesting a movement of L5 for a closed, resting or desensitized state. The competitive antagonist dihydro-β-erythroidine also induced a movement of L5 although at concentrations significantly higher than needed for current inhibition. Consequently L5, located inside the lumen of the pentamer, plays a role in both activation and inhibition of the nAChR.     

One-step purification of bacterially expressed recombinant transducin alpha-subunit and isotopically labeled PDE6 gamma-subunit for NMR analysis

Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.     

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.     

Characterization of parathyroid hormone/receptor interactions: structure of the first extracellular loop

The structural features of the first extracellular loop (ECL1) of the parathyroid hormone receptor (PTH1R) in the presence of dodecylphosphocholine micelles have been determined using high-resolution NMR techniques. The structure of the receptor fragment, PTH1R(241-285), includes three alpha-helices for residues 241-244, 256-264, and 275-284. The first and third correspond to the end and the beginning of transmembrane helices 2 and 3, respectively. Centrally located in the second helix is L(261), found to cross-link to Lys(27) of parathyroid hormone, PTH(1-34) [Greenberg, Z., Bisello, A., Mierke, D. F., Rosenblatt, M., and Chorev, M. (2000) Biochemistry 39, 8142-8152]. On the basis of nitroxide radical-induced relaxation studies, the central helix is found to associate with the surface of the membrane mimetic. These data, in conjunction with previous results indicating a preference of PTH for the lipid surface, suggest a membrane-associated pathway for the initial recognition and binding of PTH to its G-protein-coupled receptor. Using the structural features of ECL1 as determined here, along with the structure of the PTH(1-34), the intermolecular interactions consistent with the contact point between L(261)(receptor)-Lys(27)(ligand) are identified.     

Identification of functionally important cysteines in the alpha-subunit of transducin by chemical cross-linking techniques

Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, T and T. N, N-1, 2-phenylenedimaleimide (o-PDM) and N, N-1, 4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of T with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and 200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of T with both reagents produced a major species of 46-kDa. The combination of intact T and o-PDM- or p-PDM-treated T reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified T was incubated with intact T, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on T represent functionally important residues of T.     

Roles for loop 2 residues of alpha1 glycine receptors in agonist activation

The present study tested the hypothesis that several residues in Loop 2 of alpha1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50-60 on glycine responses in alpha1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a beta-turn structure of Loop 2, with odd-numbered residues in the beta-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains. We also tested the hypothesis that the charge at position 53 is important for agonist activation by measuring the glycine response of wild type (WT) and E53C GlyRs exposed to methanethiosulfonate reagents. As earlier, E53C GlyRs have a significantly higher EC(50) than WT GlyRs. Exposing E53C GlyRs to the negatively charged 2-sulfonatoethyl methanethiosulfonate, but not neutral 2-hydroxyethyl methanethiosulfonate, positively charged 2-aminoethyl methanethiosulfonate, or 2-trimethylammonioethyl methanethiosulfonate, decreased the glycine EC(50) to resemble WT GlyR responses. Exposure to these reagents did not significantly alter the glycine EC(50) for WT GlyRs. The latter findings suggest that the negative charge at position 53 is important for activation of GlyRs through its interaction with positive charge(s) in other neighboring agonist activation elements. Collectively, the findings provide the basis for a refined molecular model of alpha1GlyRs based on the recent x-ray structure of a prokaryotic pentameric ligand-gated ion channel and offer insight into the structure-function relationships in GlyRs and possibly other ligand-gated ion channels.     

Identification of monoclonal antibody 4A-binding site on the transducin alpha subunit. Immunoblotting of submaxillary Arg-C protease fragments of transducin.

Monoclonal antibody 4A (mAb 4A) against the T alpha subunit of transducin has been widely used to study the structure and function of signal transducing GTP-binding proteins involved in the regulation of visual excitation, hormonal regulation of adenylyl cyclase and ionic channels. Results of mapping the epitope-binding site of mAb 4A on T alpha have been controversial. Hamm and co-workers (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) reported that mAb 4A interacts with T alpha at the carboxyl-terminal peptide, whereas Fung and co-workers (Navon, S. E., and Fung, B. K.-k. (1988) J. Biol. Chem. 263, 489-498) showed that mAb 4A binds mainly at the amino-terminal peptide. In this report, we examine the epitope-binding site of mAb 4A by Western immunoblotting of the proteolytic fragments of T alpha generated by submaxillary Arg-C protease digestion. Submaxillary Arg-C protease cleaved T alpha at two sites, Arg-204 and Arg-310, generating two major fragments of apparent size 35 (T alpha'SM-35) and 23 kDa (T alpha'SM-23). Both fragments contain the amino-terminal peptide of T alpha but lack the carboxyl-terminal peptide. Western immunoblotting showed that mAb 4A cross-reacted with both peptides. Treatment of T alpha'SM-35 and T alpha'SM-25 with L-1-(tosylamido)-2-phenyethyl chloromethyl ketone-trypsin removed the amino-terminal 2-kDa peptide with concomitant loss of mAb 4A reactivity. This observation unequivocally confirms the result of Fung and co-workers that the epitope for mAb 4A is located on the amino-terminal 2-kDa peptide of T alpha. This conclusion should provide a more accurate interpretation of results in the literature as well as of future studies in which mAb 4A is used.     

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.     

Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.     

Phosphorescence and ODMR study of the binding interactions of acetylcholine receptor alpha-subunit peptides with alpha-cobratoxin.

Optical detection of magnetic resonance (ODMR) and phosphorescence spectroscopy have been applied to synthetic peptides derived from the alpha-subunit of the nicotinic acetylcholine receptor of Torpedo californica and their complexes with alpha-cobratoxin (CBTX). The CBTX Trp phosphorescence is strongly quenched by the proximal disulfide linkage, while the emission wavelengths and ODMR frequencies of the 18-mer alpha 181-198 indicate a more hydrophobic Trp environment than in the 12-mer alpha 185-196. Binding to CBTX produces a subtle increase in the hydrophobicity of the Trp environment for the peptides, in qualitative agreement with a recently proposed binding model, in which a receptor Trp residue interacts strongly with a hydrophobic cleft of the toxin.     

The surface of alpha-subunit loop 1 distant from the subunit interface is exposed in the hCG lutropin receptor complex

Interactions of the placental glycoprotein hormone human choriogonadotropin (hCG) with lutropin receptors (LHR) are required for maintenance of early pregnancy. Knowledge of how hCG interacts with LHR is useful for understanding the mechanism of receptor function, an issue of considerable debate. A large surface of hCG remains exposed after the hormone binds the LHR and can be readily detected with monoclonal antibodies. Here we show that the surface of hCG alpha-subunit loop 1 furthest from the beta-subunit interface can also be recognized by a monoclonal antibody when hCG is bound to the LHR. This extends the area of hCG known to be exposed in the hormone receptor complex, an observation that further restricts models of hCG-LHR interaction.     

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.     

Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex

Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.     

Domain interactions of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. alpha-FLAG or alpha-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the alpha-FLAG but not alpha-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the alpha-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor.     

Interleukin-4-specific signal transduction events are driven by homotypic interactions of the interleukin-4 receptor alpha subunit.

Interleukin-4 (IL-4) exerts its effects through a heterodimeric receptor complex (IL-4R), which contains the IL-4R(alpha) and gamma(c) subunits. IL-4R(alpha) also functions with other partner subunits in several receptor types, including the IL-13 receptor. To examine the roles of the individual subunits within IL-4R complexes, we employed a chimeric system that recapitulates native IL-4R function as verified by the activation of the kinases, JAK1 and JAK3, and induction of STAT-6. When a mutant gamma(c) subunit in which the four cytoplasmic tyrosines were converted to phenylalanine was paired with the cytoplasmic domain of the IL-4R(alpha) chain, specificity within the JAK-STAT pathway was not altered. Signaling events were examined further in cells expressing the IL-4R(alpha) chimera alone without the gamma(c) chimera. Ligand-induced homodimerization of these receptors activated the IL-4 signaling program despite the absence of gamma(c), including induction of JAK1 and STAT-6, phosphorylation of the insulin-related substrate 1 and cellular proliferation. Thus, homotypic interactions of the IL-4R(alpha) subunit are sufficient for the initiation and determination of IL-4-specific signaling events, and such interactions may be integral to signaling through IL-4R complexes.     

Role of the large cytoplasmic loop of the alpha 7 neuronal nicotinic acetylcholine receptor subunit in receptor expression and function

The role of the large intracellular loop of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in the expression of functional channels was studied. For this purpose, systematic deletions and substitutions were made throughout the loop and the ability of the mutated alpha7 subunits to support expression of functional nAChRs at the Xenopus oocyte membrane was tested. Surface nAChR expression was abolished upon removal of sequences at two regions, a 29-amino acid segment close to the N-terminus of the loop (amino acids 297-325) and adjacent to the third transmembrane region and an 11-amino acid segment near the fourth transmembrane region. Some residues (amino acids 317-322) within the 29 amino acids N-terminal segment could be substituted by others but not deleted without loss of expression, suggesting that a certain structure, determined by the number of amino acids rather than by their identity, has to be maintained in this region. The contiguous sequence M323 K324 R325 did not tolerate deletions and substitutions. Removal of the rest of the cytoplasmic loop was not deleterious; even higher expression levels (2-4-fold) were obtained upon large deletions of the loop (Delta399-432 and Delta339-370). High expression levels were observed provided that a minimal sequence of three amino acids (E371, G372, and M373) was present. In addition, some electrophysiological properties of mutant nAChRs were modified. Substitution of the EGM sequence by other protein segments produced a variety of effects, but, in general, insertions were not well tolerated, suggesting the existence of tight structural restrictions in the large cytoplasmic region of the rat alpha7 subunit.     

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.     

A basic cluster determines topology of the cytoplasmic M3-M4 loop of the glycine receptor alpha1 subunit

The inhibitory glycine receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors, which are composed of homologous subunits with four transmembrane segments (M1-M4), each. Here, we demonstrate that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop (designated M3-M4 loop) following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment.