Establishment and characterization of RNA-edited serotonin 2C receptor isoform cell models and alteration of amyloid precursor protein ectodomain secretion in HEK293 APPSwe cells

RNA editing is a mechanism for generating molecular diversity by altering the genetic code at the level of RNA. The 5-HT(2C) receptor is the only G protein-coupled receptor known to be edited. It has been reported that the non-edited 5-HT(2C) receptor stimulates secretion of the APP metabolite APP ectodomain (APPs). However, it remains unknown whether RNA-edited 5-HT(2C) receptors can also affect APPs secretion. In this study, cDNAs of five non-edited or partially/fully edited 5-HT(2C) receptor isoforms (INI, VNI, VNV, VSV and VGV) were stably transfected into HEK293APPSwe cells to detect the cell proliferation and APPs secretion. The results demonstrated that the overexpression of INI and VNI caused increased proliferation of host cells while VNV, VSV and VGV caused inverse effects (P?相似文献   

Dopamine D2 receptor isoforms expressed in AtT20 cells differentially couple to G proteins to acutely inhibit high voltage-activated calcium channels

The dopamine D2 receptor belongs to the serpentine superfamily of receptors, which have seven transmembrane segments and activate G proteins. D2 receptors are known to be linked, through Galpha(o)- and Galpha(i)-containing G proteins, to several signaling pathways in neuronal and secretory cells, including inhibition of adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs). The dopamine D2 receptor exists in two alternatively spliced isoforms, "long" and "short" (D2L, and D2S, respectively), which have identical ligand binding sites but differ by 29 amino acids in the third intracellular loop, the proposed site for G protein interaction. This has led to the speculation that the two isoforms may interact with different G proteins. We have transfected the AtT20 cell line with either D2L (KCL line) or D2S (KCS line) to facilitate experimentation on the individual isoforms. Both lines show dopamine agonist-dependent inhibition of Q-type HVA-CCs. We combined G protein antisense knock-down studies with multiwavelength fluorescence video microscopy to measure changes in HVA-CC inhibition to investigate the possibility of differential G protein coupling to this inhibition. The initial, rapid, K+ depolarization-induced increase in intracellular Ca2+ concentration is due to influx through HVA-CCs. Our studies reveal that both D2 isoforms couple to Galpha(o) to partially inhibit this influx. However, D2L also couples to Galpha(i)3, whereas D2S couples to Galpha(i)2. These data support the hypothesis of differential coupling of D2 receptor isoforms to G proteins.     

Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides

A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]gamma-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]gamma-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor-G protein interactions.     

Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors

Different isoforms of serotonin subtype 2C receptor (5-HT(2C)R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT(2C)R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An approximately 13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile156, Gly158, and Val160 (5-HT(2C)R-IGV). In contrast, the agonist was four- to five-fold less potent with 5-HT(2C)R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.     

Changes in conformation at the cytoplasmic ends of the fifth and sixth transmembrane helices of a yeast G protein-coupled receptor in response to ligand binding

The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.     

Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.     

Site-directed mutations in the third intracellular loop of the serotonin 5-HT(1A) receptor alter G protein coupling from G(i) to G(s) in a ligand-dependent manner

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.     

Contributions of intracellular loops 2 and 3 of the lutropin receptor in Gs coupling

A number of amino acids essential for Gs coupling, i.e. hot spots, were identified after in vitro Ala-scanning mutagenesis of the cytosolic extensions of helices 3, 5, and 6 and of intracellular loops 2 and 3 (IL2 and IL3) of the human LH receptor (LHR). Consistent with the results of in vitro experiments involving ligand binding and ligand-mediated signaling in transiently transfected human embryonic kidney 293 cells, computational modeling of the isolated receptor and of the receptor-G protein complexes suggests an important role of the cytosolic extension of helix 3 and the N-terminal portion of the IL2 in Gs(alpha) interaction, whereas the contribution of IL3 is marginal. Mapping the hot spots into the computational models of LHR and the LHR-Gs complexes allowed for a distinction between receptor sites required for intramolecular structural changes (i.e. I460, T461, H466, and I549) and receptor sites more likely involved in G protein recognition (i.e. R464, T467, I468, Y470, Y550, and D564). The latter sites include the highly conserved arginine of the (E/D)R(Y/W) motif, which is therefore likely to be a receptor recognition point for Gs rather than a switch of receptor activation. The results of in vitro and in silico experiments carried out in this study represent the first comprehensive delineation of functionality of the individual residues in the intracellular domains of LHR and establish potential switches of receptor activation as well as a map of the primary receptor recognition sites for Gs. A novel way to consider constitutively active mutants was inferred from this study, i.e. receptor states with improved complementarity for the G protein compared to the wild-type receptor.     

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.     

A-to-I editing of the 5HT2C receptor and behaviour.

Site-specific deamination of five adenosine residues in the pre-mRNA of the serotonin 2C receptor, 5HT2CR, alters the amino acid sequence of the encoded protein. Such RNA editing can produce 32 mRNA variants, encoding 24 protein isoforms that vary in biochemical and pharmacological properties. Because serotonin functions in the regulation of mood and behaviour, modulation of serotonin signalling by RNA editing may be relevant to such psychiatric disorders as anxiety and depression. Several recent human studies have reported changes in 5HT2CR editing in schizophrenia, major depression or suicide, but results are variable and not conclusive. Rodent studies have begun to examine effects of drug treatments and stress. Understanding the importance of 5HT2CR editing in mood and behaviour will be assisted by experiments designed to analyse multiple strains of mice, in different behavioural tests, with optimal evaluation of the time course of molecular changes.     

Development of Polyclonal Anti-D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction

Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [¹²⁵I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [¹²⁵I]-NCQ 298 bound reversibly and with high affinity (K_D= 0.14 n M ) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [¹²⁵I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to inhibit the high-affinity binding of the agonist N -propylnorapomorphine to digitonin-solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.     

Agonist-specific conformational changes in the yeast alpha-factor pheromone receptor.

The yeast alpha-factor pheromone receptor is a member of the G-protein-coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, alpha-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-factor, reduced the cleavage rate. Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist. alpha-Factor inhibited cleavage weakly at a second site near the cytoplasmic terminus of the seventh transmembrane helix, whereas the antagonist showed a stronger inhibition of cleavage at this site and at another site in the C-terminal domain of the receptor. The alpha-factor-induced conformational changes appeared to be inherent properties of the receptor, as they were retained in G-protein-deficient mutants. Moreover, a mutant receptor (ste2-L236H) that affects the third loop and is defective for G-protein coupling retained the ability to undergo the agonist-induced conformational changes. These results are consistent with a model in which G-protein activation is limited by the availability of specific contacts between the G protein and the third intracellular loop of the receptor. The antagonist appears to promote a distinct conformational state that differs from either the unoccupied or the agonist-occupied state.     

Active peptidic mimics of the second intracellular loop of the V(1A) vasopressin receptor are structurally related to the second intracellular rhodopsin loop: a combined 1H NMR and biochemical study

Vasopressin (VP) receptors belong to the widespread G protein-coupled receptor family. The crucial role of VP receptor intracellular loops in the coupling with the heterotrimeric G proteins was previously demonstrated by construction of a vasopressin receptor chimera. Yet, no fine structural data are available concerning the receptor molecular determinants involved in their interactions with G proteins. In this study, we synthesized both a linear and a cyclic form of the second intracellular loop (i2) of the human V(1a) vasopressin receptor isoform that is important for the interaction between the alphaq/alpha11 G protein and the receptor. These two peptides are biologically active. They specifically inhibit vasopressin binding to the V(1a) receptor, suggesting that the corresponding endogenous peptides contribute to the structure of the vasopressin binding site via intra- or intermolecular interactions with the core of the V(1a) receptor. The i2 peptide structures were determined by (1)H NMR. Both exhibit a helix and helical elements in their N- and C-terminal parts, respectively, separated by a turn imposed by a proline residue. More interestingly, the central Pro-Leu motif conserved in many GPCRs and thought to be important for coupling to G proteins can adopt different conformations. The "U" shape structure of the i2 loop is compatible with its anchoring to transmembrane domains III and IV and is very similar to the shape of bovine rhodopsin i2. Altogether, these data contribute to a better understanding of the structure of a not yet crystallized GPCR using the mimetic peptide approach.     

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.