细胞受体与疾病的相关性研究进展

受体是存在于效应细胞中的重要功能蛋白，它和配体具有高亲和力，受体异常亦称受体病，拟就近年来细胞受体和受体病的研究进展作一综述。     

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.     

Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.     

褪黑素受体

褪黑素是松果体分泌的主要激素,其功能活动通过特异的Ｇ蛋白耦联受体介导,本文综述褪黑素受体 分布、药理学特性,受体的克隆及受体基因结构特点。     

Dopamine D4 receptor oligomerization--contribution to receptor biogenesis

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.2)R, D(4.4)R and D(4.7)R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D(4)R biogenesis and quality control in the endoplasmic reticulum plays an important role for D(4)R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D(4.4)R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D(4)R. The different polymorphic repeat variants of the D(4)R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D(4)Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.     

Molybdate-stabilized glucocorticoid receptor: evidence for a receptor heteromer

The composition of the molybdate-stabilized glucocorticoid receptor (GR) complex has been investigated with a monoclonal antibody against the steroid-binding Mr 94 000 (94K) GR protein. It was concluded that one antibody molecule binds one 94K GR molecule. This finding constituted the basis for calculating the number of antibodies bound to the molybdate-stabilized nonactivated GR complex, which has an Mr of 302 000 (302K). Gel filtration on Sephacryl S-400 and density gradient centrifugation showed that only one antibody molecule bound to the molybdate-stabilized GR complex (calculated relative molecular mass for the antibody--molybdate-stabilized GR complex, 456 000; relative molecular mass for one antibody molecule, 157 000). Furthermore, experiments performed with a second antibody immunoprecipitation assay in the presence of an excess of both antibody and GR confirmed the above results. The possibility of steric hindrance not allowing more than one antibody molecule to bind to the molybdate-stabilized GR complex could be excluded. These results suggest that the molybdate-stabilized GR complex with an Mr of 302K only contains one steroid-binding 94K GR molecule and therefore represents a heteromeric complex.     

Novel receptor partners and function of receptor activity-modifying proteins

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.     

Apolipoprotein E receptor 2 interactions with the N-methyl-D-aspartate receptor

In our previous studies we showed that apoE treatment of neurons activated ERK 1/2 signaling, and activation was blocked by treatment with inhibitors of the low density lipoprotein receptor family, the N-methyl-d-aspartate (NMDA) receptor antagonist MK 801, and calcium channel blockers. We hypothesized an interaction between the low density lipoprotein receptor family members and the NMDA receptor. In the present study, we confirmed through co-immunoprecipitation experiments an interaction between the apoE receptor, ApoEr2, and NMDAR1 through their extracellular domains. We also found that the PDZ1 domain of PSD95, a postsynaptic scaffolding protein, interacted with the C terminus of ApoEr2 via an alternatively spliced, intracellular exon. This interaction between ApoEr2 and PSD95 in neurons was modulated by NMDA receptor activation and an ApoEr2 ligand. We also found that the PDZ2 domain of PSD95 interacted with the NR2A and NR2B subunits of NMDA receptors. Full-length PSD95 increased cell surface levels of ApoEr2 and its cleavage, resulting in increases in secreted ApoEr2 and C-terminal fragments of ApoEr2. These studies suggest that ApoEr2 can form a multiprotein complex with NMDA receptor subunits and PSD95.     

The mobile receptor hypothesis for cell membrane receptor action

The mobile receptor hypothesis proposes that membrane receptors diffuse within the plane of the membrane and when occupied with ligand reversibly, associate with membrane enzymes or channels regulating their activity. Some of the implications and evidence for this model are presented.     

Coupling of agonist-induced AMPA receptor internalization with receptor recycling

Excitatory post-synaptic currents in the CNS are primarily mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in response to glutamate. Internalization of cell-surface receptors has been shown to be one mechanism by which to control receptor function. To test for agonist control of AMPA receptor plasma membrane expression we used biochemical assays to study AMPA receptor internalization and insertion processes. In heterologous cells, we observed a slow constitutive internalization and a rapid agonist-induced internalization of AMPA receptors. To our surprise, however, agonist treatment had no effect on the steady-state levels of AMPA receptors on the cell surface. To examine whether this could be explained by an agonist-induced increase in the insertion rate of AMPA receptors into the plasma membrane we developed an assay to independently measure receptor insertion. Remarkably, agonist treatment of cells also dramatically increased AMPA receptor plasma membrane insertion rates. In addition, using an assay to measure recycling of internalized pools we found that internalized receptors are rapidly recycled to the cell surface. These results suggest that agonist-induced receptor internalization is coupled to increases in receptor recycling. This increase in receptor flux through intracellular pools may allow for rapid changes in receptor surface expression by independent regulatory control of internalization and insertion.     

Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in adipogenesis. PPARgamma binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARgamma-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARgamma is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARgamma. Here we report that PPARgamma and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARgamma-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARgamma. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARgamma- and RXR-specific ligands may occur at the level of selective coactivator recruitment.     

Estrogen receptor stereochemistry: receptor binding and hormonal responses

Estrogen stimulation of the uterus elicits a spectrum of biochemical responses which are customarily linked together. DES and certain structural analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (I), and pseudo DES (PD), were used as probes to segregate various genomic responses previously considered interrelated, most notably the events of specific protein synthesis, DNA synthesis, and mitosis. These compounds have poor uterotrophic activity; except for I, they interact specifically with mouse uterine estrogen receptors (ER) with high affinity. All translocate stoichiometrically similar amounts of ER complex to the nucleus. IA and IB possess a single chiral carbon atom and exist as a mixture of enantiomers (ENT). We investigated whether the poor biological activity of IA could be explained by differential activity of the enantiomers. The IA ENT were separated to greater than 98% purity using a chirally active HPLC column. Competitive binding assays to cytosolic ER demonstrated a stereochemical chiral preference. This preference was also evident from nuclear ER translocation experiments. IB was as active as DES to induce mouse uterine glucose 6-phosphate dehydrogenase (G-6-PD), while the other compounds had weak activity. Induction of cytosolic progesterone receptor (PR) was stimulated by all the DES compounds. Ornithine decarboxylase (ODC) was stimulated 600% by DES and 180% by IB; the other compounds had no significant activity. Uterine DNA synthesis was increased by DES and IB. Thymidine autoradiography indicated nuclear labeling was occurring primarily in luminal epithelium. Treatment with PD increased uterine cell height but not cell numbers, suggesting the two responses are not necessarily interdependent as previously thought and may require two separate receptor interactions. Such a probe should be useful in studying the individual events involved in estrogen-induced uterine growth. These data also indicate that induction of ER, PR and G-6-PD are not coupled. Therefore, stimulation of a certain uterine response may depend on the structure of the particular ligand receptor complex formed, and its interaction may be regulated by specificity at the genomic acceptor site.     

Acetylcholine receptor: effects of proteolysis on receptor metabolism

Previous studies (Miskin, R., T. G. Easton, and E. Reich, 1970, Cell. 15:1301-1312) have shown that sarcoma virus transformation and tumor promoters reduced the cell surface concentration of acetylcholine receptors (AChR) in differentiating chick embryo myogenic cultures. Both of these agents also induced high rates of plasminogen activator (PA) synthesis in myogenic cultures (Miskin, R., T. G. Easton, A. Maelicke, and E. Reich, 1978, Cell. 15:1287-1300), and the present work was performed to establish whether proteolysis might significantly affect receptor metabolism. Proteolysis in myogenic cultures was modulated by one or more of the following: stimulation of PA synthesis, direct addition of plasmin, removal of plasminogen, or addition of plasmin inhibitors. The results were: (a) When the rates of proteolysis were raised either by addition of plasmin or by stimulating PA synthesis in the presence of plasminogen, both the steady-state concentration and the half-life of surface AChR decreased, but the rate of receptor synthesis was unaffected. (b) The magnitude of these effects, and their dependence on added plasminogen, indicated that proteolysis initiated by plasminogen activation could account almost entirely for the reduction in receptor half-life produced by sarcoma virus transformation and phorbol ester. (c) The rate of receptor synthesis, which is also reduced by viral transformation and tumor promoters, was not modified by proteolysis; hence plasmin action may be responsible for a large part, but not all of the change in surface receptor under these conditions. (d) The plasmin catalysed changes in receptor parameters appear to occur in response to modified membrane metabolism resulting from proteolysis of surface components other than AChR itself.     

Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization

Extracellular calcium rapidly controls PTH secretion through binding to the G protein-coupled calcium-sensing receptor (CASR) expressed in parathyroid glands. Very little is known about the regulatory proteins involved in desensitization of CASR. G protein receptor kinases (GRK) and beta-arrestins are important regulators of agonist-dependent desensitization of G protein-coupled receptors. In the present study, we investigated their role in mediating agonist-dependent desensitization of CASR. In heterologous cell culture models, we found that the transfection of GRK4 inhibits CASR signaling by enhancing receptor phosphorylation and beta-arrestin translocation to the CASR. In contrast, we found that overexpression of GRK2 desensitizes CASR by classical mechanisms as well as through phosphorylation-independent mechanisms involving disruption of Galphaq signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in beta-arrestin2 null mice, suggesting a functional role of beta-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and beta-arrestins play key roles in regulating CASR responsiveness in parathyroid glands.     

Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation

Given that ligand binding is essential for the rapid internalization of epidermal growth factor receptor (EGFR), the events induced by ligand binding probably contribute to the regulation of EGFR internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and autophosphorylation. Whereas the initial results are controversial regarding the role of EGFR kinase activity in EGFR internalization, more recent data suggest that EGFR kinase activation is essential for EGFR internalization. However, we have shown here that inhibition of EGFR kinase activation by mutation or by chemical inhibitors did not block EGF-induced EGFR internalization. Instead, proper EGFR dimerization is necessary and sufficient to stimulate EGFR internalization. We conclude that EGFR internalization is controlled by EGFR dimerization, rather than EGFR kinase activation. Our results also define a new role for EGFR dimerization: by itself it can drive EGFR internalization, independent of its role in the activation of EGFR kinase.