Molecular cloning and characterization of crustacean type-one dopamine receptors: D1alphaPan and D1betaPan

Dopamine (DA) differentially modulates identified neurons in the crustacean stomatogastric nervous system (STNS). While the electrophysiological actions of DA have been well characterized, little is known about the dopaminergic transduction cascades operating in this system. As a first step toward illuminating the molecular underpinnings of dopaminergic signal transduction in the crustacean STNS, we have cloned and characterized two type-one DA receptors (DARs) from the spiny lobster (Panulirus interruptus): D(1alphaPan) and D(1betaPan). We found that the structure and function of these arthropod DARs are well conserved across species. Using a heterologous expression system, we determined that DA, but not serotonin, octopamine, tyramine or histamine activates these receptors. When stably expressed in HEK cells, the D(1alphaPan) receptor couples with Gs, and DA elicits an increase in [cAMP]. The D(1betaPan) receptor responds to DA with a net increase in [cAMP] that is mediated by Gs and Gz.     

Extracellular cAMP inhibits D1 dopamine receptor expression in CAD catecholaminergic cells via A2a adenosine receptors

The expression of D1 dopamine (DA) receptor gene is regulated during development, aging, and pathophysiology. The extracellular factors and signaling mechanisms that modulate the expression of D1 DA receptor have not been well characterized. Here, we present novel evidence that endogenous D1 DA receptor expression is inhibited by extracellular cAMP in the Cath.A Derived (CAD) catecholaminergic neuronal cell line. CAD cells express the multi-drug resistance protein 5 transporters and secrete cAMP. Addition of exogenous cAMP decreases D1 receptor mRNA and protein greater than fourfold in 24 h. The cAMP-induced decrease of D1 receptor mRNA levels is blocked by cGMP and by 1,3-dipropyl-8-(p-sulfo-phenyl)xanthine, an inhibitor of ecto-phosphodiestrase. Extracellular AMP, a metabolite of cAMP, also independently decreased D1 receptor mRNA levels. Inhibitors of ecto-nucleotidases, alpha,beta-methyleneadenosine 5'-di-phosphate and GMP, completely blocked the decrease of D1 receptor mRNA by extracellular cAMP, but only partially blocked the decrease induced by extracellular AMP. Levamisole, an inhibitor of tissue non-specific alkaline phosphatase, completely blocked the AMP-induced decrease of D1 receptor mRNA. The extracellular cAMP, AMP, and adenosine (ADO)-induced decrease in D1 receptor mRNA expression are mediated by A2a ADO receptor subtype. The results suggest a novel molecular mechanism linking activation of A2a ADO receptors with inhibition of D1 DA receptor expression.     

beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.     

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.     

IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi

Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N(G)-monomethyl-l-arginine). Hence, insulin might interfere with G(i). Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i).     

Involvement of both G(q/11) and G(s) proteins in gonadotropin-releasing hormone receptor-mediated signaling in L beta T2 cells

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptor-mediated signaling in L beta T2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LH beta genes in these cells. Signaling via the G(i) subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LH beta induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters G beta gamma-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of G alpha(q) (Q209L) and was blocked by a cell-permeable peptide that uncouples G alpha(q) from GPCRs. Furthermore, chronic activation of G alpha(q) signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples G alpha(s) from receptors was also able to inhibit ERK, c-Fos, and LH beta, indicating that both G(q/11) and G(s) proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on G(s) and G(q/11) and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of G alpha(q) (Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both G(q) and G(s) signaling to regulate gene expression in L beta T2 cells.     

Modulation of network pacemaker neurons by oxygen at the anaerobic threshold

Previous in vitro and in vivo studies showed that the frequency of rhythmic pyloric network activity in the lobster is modulated directly by oxygen partial pressure (PO(2)). We have extended these results by (1) increasing the period of exposure to low PO(2) and by (2) testing the sensitivity of the pyloric network to changes in PO(2) that are within the narrow range normally experienced by the lobster (1 to 6?kPa). We found that the pyloric network rhythm was indeed altered by changes in PO(2) within the range typically observed in vivo. Furthermore, a previous study showed that the lateral pyloric constrictor motor neuron (LP) contributes to the O(2) sensitivity of the pyloric network. Here, we expanded on this idea by testing the hypothesis that pyloric pacemaker neurons also contribute to pyloric O(2) sensitivity. A 2-h exposure to 1?kPa PO(2), which was twice the period used previously, decreased the frequency of an isolated group of pacemaker neurons, suggesting that changes in the rhythmogenic properties of these cells contribute to pyloric O(2) sensitivity during long-term near-anaerobic (anaerobic threshold, 0.7-1.2?kPa) conditions.     

8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., D?skeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.     

A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.     

Dynamic regulation, desensitization, and cross-talk in discrete subcellular microdomains during beta2-adrenoceptor and prostanoid receptor cAMP signaling

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.     

The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein beta arrestin. Here we report that this activating pathway is independent of G alpha s, G alpha i, G alpha q or G betagamma and that the V2R-mediated activation of G alpha s inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the beta arrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and beta arrestin prevents signal propagation to the nucleus. Beta arrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the beta arrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that beta arrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.     

Specific combinations of G-protein subunits discriminate hormonal signalling in rat pituitary (GH(3)) cells in culture

It was previously shown that hormone receptor coupling to voltage-dependent calcium channels in prolactin and growth hormone-producing GH(3) cells was heavily dependent on the specific heterotrimeric combinations of alpha, beta, and gamma subunits of the guanosine triphosphate (GTP)-binding protein family. Consequently, we assessed whether this was also the case for hormonal modulation of the adenylate cyclase (AC) and phospholipase C (PL-C) effector enzymes in GH(3) cells in culture. By employing polyclonal antibodies directed towards C-terminal decapeptides of various alpha subunits in membrane assays, as well as antisense oligonucleotides towards certain beta- and gamma-subunit genes in whole-cell incubations, it was possible to unravel a tentative profile of heterotrimers preferred by some of the seven-transmembrane-stretch receptors in their modulation of AC and PL-C activities. Vasoactive intestinal peptide (VIP) and thyroliberin (TRH) activate membrane-bound AC through alpha(s)beta(2)gamma(2), while somatostatin (SRIH) and dopamine (DA) inhibited the AC through alpha(i2)beta(1)gamma(3). TRH activated membrane-bound PL-C through alpha(q/11)beta(4)gamma(2), while DA inhibition of the PL-C was accomplished via alpha(o)beta(3)gamma(4). Hence, it seems that not only the specificity of alpha subunits determines the coupling between G protein-associated receptors in GH cells, the receptor binding to G proteins also requires certain combinations of beta and gamma subunits.     

Peptide sequences from the hypervariable regions of two monoclonal anti-idiotypic antibodies against the thyrotropin (TSH) receptor are similar to TSH and inhibit TSH-increased cAMP production in FRTL-5 thyroid cells.

Monoclonal antibodies, D2 and 4G11, selected by the autoantiidiotypic approach following injection of thyrotropin (TSH) into mice, mimic TSH in binding to receptors on thyroid membranes. Based on TSH receptor transfection studies, D2 and 4G11 show unequivocal specificity for the TSH receptor. To see if the complementary determining regions (CDRs) of these antibodies share any primary sequence similarities to regions of TSH critical for receptor binding, we deduced the primary structure of the variable regions of D2 and 4G11 by sequencing the immunoglobulin mRNA. We found that CDR1 of 4G11K and CDR2 of D2 mu show sequence similarity to regions of TSH alpha and TSH beta that had been previously implicated in the interaction of the hormone with its receptor. We tested the inhibitory effects of synthetic peptides from D2 mu-CDR2 and 4G11K-CDR1 on the binding of the corresponding antibodies to rat thyroid FRTL-5 cells and found an EC50 of 0.1 and 1 microM, respectively. TSH-derived peptides with similarity to D2 mu-CDR2 and 4G11K-CDR1 showed a significant but lesser effect on the binding of 4G11 or D2 to thyroid cells. Additionally, we tested the effects of the CDR peptides and TSH-derived peptides on TSH-stimulated cAMP production in FRTL-5 cells and found that D2 mu-CDR2 and 4G11K-CDR1 inhibited this activity, D2 mu-CDR2 most strongly (EC50 10 microM). Thus, linear sequences from the CDRs of these autoantiidiotypic antibodies with similarity to sequences from both subunits of TSH appear to interact with the TSH receptor. These data support previous studies indicating the complexity of the interaction between TSH and its receptor and advance earlier findings that such immunologic approaches are useful in dissecting receptor-ligand interactions.     

Stable association of G proteins with beta 2AR is independent of the state of receptor activation.

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.     

Differential coupling of beta3A- and beta3B-adrenergic receptors to endogenous and chimeric Galphas and Galphai

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of G(alpha)q with the COOH-terminal heptapeptide of G(alpha)s or G(alpha)i were used to assess the relative coupling of beta(3)-adrenergic receptor (beta(3)-AR) splice variants (beta(3A) and beta(3B)) to G(alpha)s and G(alpha)i. The G(alpha)q/s and G(alpha)q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca(2+)(i)). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat beta(3)-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, beta(3)-AR-induced coupling to G(alpha)q/s produced a rapid eightfold increase in Ca(2+)(i) followed by a slow decay to levels 25% above baseline. G(alpha)q/i also linked rat beta(3)-AR to mobilization of Ca(2+)(i) in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca(2+)(i) was only 30% of the response obtained with G(alpha)q/s. Activation of the rat beta(3)-AR also increased GTP binding to endogenous G(alpha)i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse beta(3A)- and beta(3B)-AR to G(alpha)i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to G(alpha)q/s and to increases in intracellular cAMP through endogenous G(alpha)s. The beta(3A)- and beta(3B)-AR coupled equivalently to G(alpha)q/i, but the temporal patterns of Ca(2+)(i) mobilization indicated that coupling was significantly less efficient than coupling to G(alpha)q/s. Collectively, these findings indicate less efficient but equivalent coupling of beta(3A)- and beta(3B)-AR to G(alpha)i vs. G(alpha)s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to beta(3)-AR activation.     

Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.     

Regulation of guanylate cyclase by a guanine nucleotide binding protein, G alpha 2, in Dictyostelium discoideum.

Binding of cyclic AMP (cAMP) to the cell surface receptor induces a transient activation of guanylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) which lacks G alpha 2, a guanine nucleotide binding protein, does not respond to cAMP. We found that 2,3-dimercapto-1-propanol (BAL) induced a continuous activation both in the frigid and in its parents. Therefore, the BAL-induced continuous activation of guanylate cyclase is independent of G alpha 2. We also found that cAMP enhanced the BAL-induced continuous activation in the frigid mutant. This result suggests that an unidentified signal transduction mechanism from the cAMP-receptor besides the one involving G alpha 2 plays a role in the enhancement of activation. Lastly, we found that the BAL-induced continuous activation was terminated by cAMP in the parental strain, but not in the frigid mutant. Therefore, the cAMP-induced suppression on the BAL-induced continuous activation is mediated through G alpha 2.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

Crustacean dopamine receptors: localization and G protein coupling in the stomatogastric ganglion

Neuromodulators, such as dopamine (DA), control motor activity in many systems. To begin to understand how DA modulates motor behaviors, we study a well-defined model: the crustacean stomatogastric nervous system (STNS). The spiny lobster STNS receives both neuromodulatory and neurohormonal dopaminergic input, and extensive background information exists on the cellular and network effects of DA. However, there is a void of information concerning the mechanisms of DA signal transduction in this system. In this study, we show that Gs, Gi, and Gq are activated in response to DA in STNS membrane preparations from five crustacean species representing distant clades in the order Decapoda. Three evolutionarily conserved DA receptors mediate this response in spiny lobsters: D_1αPan, D_1βPan and D_2αPan. G protein coupling for these receptors can vary with the cell type. In the native membrane, the D_1αPan receptor couples with Gs and Gq, the D_1βPan receptor couples with Gs, and the D_2αPan receptor couples with Gi. All three receptors are localized exclusively to the synaptic neuropil and most likely generate global biochemical signals that alter ion channels in distant compartments, as well as local signals.     

Influence of differential stability of G protein βγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-β

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.