Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.     

Crystal structure of the PDZ1 domain of human Na(+)/H(+) exchanger regulatory factor provides insights into the mechanism of carboxyl-terminal leucine recognition by class I PDZ domains

The Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50) contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. The NHERF PDZ1 domain interacts specifically with the motifs DSLL, DSFL, and DTRL present at the carboxyl termini of the beta(2) adrenergic receptor (beta(2)AR), the platelet-derived growth factor receptor (PDGFR), and the cystic fibrosis transmembrane conductance regulator (CFTR), respectively, and plays a central role in the physiological regulation of these proteins. The crystal structure of the human NHERF PDZ1 has been determined at 1.5 A resolution using multiwavelength anomalous diffraction phasing. The overall structure is similar to known PDZ structures, with notable differences in the NHERF PDZ1 carboxylate-binding loop that contains the GYGF motif, and the variable loop between the beta2 and beta3 strands. In the crystalline state, the carboxyl-terminal sequence DEQL of PDZ1 occupies the peptide-binding pocket of a neighboring PDZ1 molecule related by 2-fold crystallographic symmetry. This structure reveals the molecular mechanism of carboxyl-terminal leucine recognition by class I PDZ domains, and provides insights into the specificity of NHERF interaction with the carboxyl termini of several membrane receptors and ion channels, including the beta(2)AR, PDGFR, and CFTR.     

The PDZ binding motif of the beta 1 adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes

Beta(1) and beta(2) adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of beta(1)ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of beta(2)ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The beta(2)AR undergoes agonist-induced endocytosis in cardiac myocytes while the beta(1)AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of beta(1)AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the beta(1)AR (beta(1)AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of beta(1)AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the beta(2)AR. The secondary decrease in the contraction rate was mediated by G(i) and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type beta(2)AR and the mutated beta(1)AR-PDZ, and blocked the coupling of both receptors to G(i). Finally, treating myocytes with a membrane-permeable peptide representing beta(1)AR PDZ motif caused the endogenous beta(1)AR to behave like beta(1)AR-PDZ. These studies suggest that association of the beta(1)AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G(i).     

Structural basis of beta-adrenergic receptor function

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.     

Characterization of the monomeric form of the transmembrane and cytoplasmic domains of the integrin beta 3 subunit by NMR spectroscopy

We have characterized a membrane protein containing residues P688-T762 of the integrin beta3 subunit, encompassing its transmembrane and cytoplasmic domains, by nuclear magnetic resonance spectroscopy. Under conditions in which it is monomeric in dodecylphosphocholine micelles, the protein consists mainly of alpha-helical structures. An amino-terminal helix corresponding to the beta3 transmembrane helix extends into the membrane-proximal region of the cytoplasmic domain. Moreover, following an apparent hinge at residues H722-D723, residues K725-A735 are mostly alpha-helical. In the presence of membrane-mimicking detergents, the cytoplasmic domain connected to the transmembrane helix is substantially ordered at pH 4.8 and 50 degrees C. Its carboxyl-terminal end takes on a turn-helix configuration characteristic of the immunoreceptor tyrosine-based activation motif. These structural features of the beta3 subunit should help to explain its interaction with numerous cytosolic interacting proteins and begin to illuminate the mechanism of integrin activation.     

Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors

In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.     

Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting

Although homodimerization has been demonstrated for a large number of G protein-coupled receptors (GPCRs), no general role has been attributed to this process. Because it is known that oligomerization plays a key role in the quality control and endoplasmic reticulum (ER) export of many proteins, we sought to determine if homodimerization could play such a role in GPCR biogenesis. Using the beta2-adrenergic receptor (beta2AR) as a model, cell fractionation studies revealed that receptor homodimerization is an event occurring as early as the ER. Supporting the hypothesis that receptor homodimerization is involved in ER processing, beta2AR mutants lacking an ER-export motif or harboring a heterologous ER-retention signal dimerized with the wild-type receptor and inhibited its trafficking to the cell surface. Finally, in addition to inhibiting receptor dimerization, disruption of the putative dimerization motif, 276GXXXGXXXL284, prevented normal trafficking of the receptor to the plasma membrane. Taken together, these data indicate that beta2AR homodimerization plays an important role in ER export and cell surface targeting.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.     

Mutational analysis of cell cycle inhibition by integrin beta1C

Integrin beta1C is an alternatively spliced cytoplasmic variant of the beta1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by beta1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of beta1C (residues 732-805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the beta1A, beta1B, beta1D, beta3, and beta5 cytoplasmic domains had weak and variable effects. The beta1C cytoplasmic domain is composed of a membrane proximal region (732-757) common to all beta1 variants and a COOH-terminal 48-amino acid domain (758-805) unique to beta1C. The beta1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by beta1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the beta1C-specific domain showed that the 18-amino acid sequence 775-792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally, beta1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/beta1C (758-805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for beta1C as a growth inhibitor in vivo.     

Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues

Nerve growth factor (NGF) is a polypeptide important for normal development of the nervous system and promotion of survival and differentiation of sensory and sympathetic neurons in culture. The cellular effects of NGF are mediated by a specific cell surface molecule, nerve growth factor receptor (NGF-R). In the present study we have used a monoclonal antibody against human NGF-R to examine, by the avidin-biotin-immunoperoxidase method, the receptor distribution in a wide range of normal tissues and in more than 200 malignant tumors. Our results show that (a) human NGF-R is expressed in the peripheral nervous system but not in any of the central nervous system areas tested; (b) NGF-R expression is not restricted to neural tissues but is also found in a number of normal epithelial, mesenchymal, and lymphoid tissues; (c) NGF-R expression changes during normal development; and (d) NGF-R expression in malignant tumors generally parallels its normal tissue distribution. Thus, NGF-R is detected in a proportion of neuroectoderm-derived tumors, carcinomas, and lymphomas, and also in a characteristic group of small round-cell tumors (Ewing's sarcomas and embryonal rhabdomyosarcomas). These findings suggest a normal regulatory role for NGF in both neuronal and non-neuronal cells and identify a range of human tumors in which the NGF/NGF-R system may contribute to the malignant phenotype.     

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.     

Syntrophins regulate alpha1D-adrenergic receptors through a PDZ domain-mediated interaction

To find novel cytoplasmic binding partners of the alpha1D-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha1D-AR C terminus as bait was performed on a human brain cDNA library. Alpha-syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an alpha1D-AR-interacting protein. Alpha-syntrophin specifically recognized the C terminus of alpha1D- but not alpha1A- or alpha1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta1, and beta2 but not gamma1 or gamma2 showed strong selective interactions with the alpha1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha1D- but not alpha1A- or alpha1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha1D-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of alpha1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.     

Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis.

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.     

Integrin activation involves a conformational change in the alpha 1 helix of the beta subunit A-domain

The ligand-binding region of integrin beta subunits contains a von Willebrand factor type A-domain: an alpha/beta "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the betaA-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the beta(1) A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). The ability of Mn(2+) and Mg(2+) to stimulate 12G10 binding is abrogated by mutation of the MIDAS motif, demonstrating that the MIDAS is a Mn(2+)/Mg(2+) binding site and that occupancy of this site induces conformational changes in the A-domain. The cation-regulated region of the 12G10 epitope maps to Arg(154)/Arg(155) in the alpha1 helix. Our results demonstrate that the alpha1 helix undergoes conformational alterations during integrin activation and suggest that Mn(2+) acts as a potent activator of beta(1) integrins because it can promote a shift in the position of this helix. The mechanism of beta subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin alpha subunits.     

大鼠基底前脑NGF-R及CHAT免疫反应阳性神经元的分布:免疫双标法

本文用免疫组化双标法观察了神经生长因子受体(NGF-R)及胆碱乙酰转移酶(ChAT)免疫反应阳性神经元在成鼠基底前脑内的分布,结果发现嗅结节、隔内侧核、斜角带核、腹侧苍白球及基底大细胞核均有NGF-R及ChAT免疫反应阳性神经元.免疫组化双标染色发现,大部分免疫反应阳性神经元的NGF-R与ChAT共存,部分神经元呈单纯NGF-R或ChAT阳性,但这种NGF-R和ChAT的共存情况在不同区域不完全相同.在隔内侧核和斜角带核,大多数的NGF-R阳性神经元和ChAT阳性神经元共存,但在腹侧仓白球和基底大细胞核,两者共存的神经元较前两区为少.此外ChAT阳性神经元在尾壳核中分布较均匀,而NGF-R阳性神经元较少见.研究结果表明,大多数胆碱能神经元有NGF-R,提示NGF对胆碱能神经元的保护和激活作用,部分可能是通过直接与NGF受体的结合而发生作用.     

3D structure and significance of the GPhiXXG helix packing motif in tetramers of the E1beta subunit of pyruvate dehydrogenase from the archeon Pyrobaculum aerophilum.

As part of a structural genomics project, we have determined the 2.0 A structure of the E1beta subunit of pyruvate dehydrogenase from Pyrobaculum aerophilum (PA), a thermophilic archaeon. The overall fold of E1beta from PA is closely similar to the previously determined E1beta structures from humans (HU) and P. putida (PP). However, unlike the HU and PP structures, the PA structure was determined in the absence of its partner subunit, E1alpha. Significant structural rearrangements occur in E1beta when its E1alpha partner is absent, including rearrangement of several secondary structure elements such as helix C. Helix C is buried by E1alpha in the HU and PP structures, but makes crystal contacts in the PA structure that lead to an apparent beta(4) tetramer. Static light scattering and sedimentation velocity data are consistent with the formation of PA E1beta tetramers in solution. The interaction of helix C with its symmetry-related counterpart stabilizes the tetrameric interface, where two glycine residues on the same face of one helix create a packing surface for the other helix. This GPhiXXG helix-helix interaction motif has previously been found in interacting transmembrane helices, and is found here at the E1alpha-E1beta interface for both the HU and PP alpha(2)beta(2) tetramers. As a case study in structural genomics, this work illustrates that comparative analysis of protein structures can identify the structural significance of a sequence motif.