Dominant negative mutants of filamin A block cell surface expression of the D2 dopamine receptor

Protein interaction screens have revealed an interaction between the D2 dopamine receptor and the actin cross-linking protein filamin A. However, the physiological significance of this interaction has not been explained. To better understand the role of filamin A in D2 receptor-mediated signaling, we examined the effect of disrupting filamin A/D2 receptor interaction. Overexpression of a truncated form of filamin A (repeat units 18-19 containing the D2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface D2 receptors. These results suggest that disruption of the linkage between D2 receptors and the actin cytoskeleton destabilizes plasma membrane-associated D2 receptors. Several missense mutations within repeat unit 19 of filamin A were identified that abrogate filamin A/D2 receptor interaction. Introduction of mutant and wild-type filamin A into filamin A-deficient M2 cells demonstrated that wild-type filamin A, but not the filamin A-binding mutants, was able to promote cell-surface expression of D2 receptors. Together, these studies provide evidence that filamin A/D2 receptor interaction is required for the proper targeting or stabilization of D2 dopamine receptors at the plasma membrane.     

G protein activation by human dopamine D3 receptors in high-expressing Chinese hamster ovary cells: A guanosine-5'-O-(3-[35S]thio)- triphosphate binding and antibody study

Despite extensive study, the G protein coupling of dopamine D3 receptors is poorly understood. In this study, we used guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]-GTPgammaS) binding to investigate the activation of G proteins coupled to human (h) D3 receptors stably expressed in Chinese hamster ovary (CHO) cells. Although the receptor expression level was high (15 pmol/mg), dopamine only stimulated G protein activation by 1.6-fold. This was despite the presence of marked receptor reserve for dopamine, as revealed by Furchgott analysis after irreversible hD3 receptor inactivation with the alkylating agent, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline). Thus, half-maximal stimulation of [35S]-GTPgammaS binding required only 11.8% receptor occupation of hD3 sites. In contrast, although the hD2(short) receptor expression level in another CHO cell line was 11-fold lower, stimulation by dopamine was higher (2.5-fold). G protein activation was increased at hD3 and, less potently, at hD2 receptors by the preferential D3 agonists, PD 128,907 [(+)-(4aR,10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H- [1]benzopyrano[4,3-b]-1, 4-oxazin-9-ol] and (+)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin). Furthermore, the selective D3 antagonists, S 14297 ((+)-[7-(N, N-dipropylamino)-5,6,7, 8-tetrahydro-naphtho(2,3b)dihydro-2,3-furane]) and GR 218,231 (2(R, S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4- tetrahydronaphtalene), blocked dopamine-stimulated [35S]GTPgammaS binding more potently at hD3 than at hD2 sites. Antibodies against Galphai/alphao reduced dopamine-induced G protein activation at both CHO-hD3 and -hD2 membranes, whereas GalphaS antibodies had no effect at either site. In contrast, incubation with anti-Galphaq/alpha11 antibodies, which did not affect dopamine-induced G protein activation at hD2 receptors, attenuated hD3-induced G protein activation. These data suggest that hD3 receptors may couple to Galphaq/alpha11 and would be consistent with the observation that pertussis toxin pretreatment, which inactivates only Gi/o proteins, only submaximally (80%) blocked dopamine-stimulated [35S]GTPgammaS binding in CHO-hD3 cells. Taken together, the present data indicate that 1) hD3 receptors functionally couple to G protein activation in CHO cells, 2) hD3 receptors activate G proteins less effectively than hD2 receptors, and 3) hD3 receptors may couple to different G protein subtypes than hD2 receptors, including nonpertussis sensitive Gq/11 proteins.     

Roles of conserved intracellular sequences regions for the proper expression of dopamine D2 and D3 receptors

The dopamine D(2) receptor and D(3) receptor (D(2)R, D(3)R) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the D(3)R signals are 2 approximately 5 times less efficient than D(2)R. Previous studies have suggested that conformational constraints of D(3)R might be responsible for the poor coupling with the G protein. To this hypothesis, point mutations were introduced into some of the conserved regions between D(2)R and D(3)R, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the 1(st) intracellular loop, SS motif in the 2(nd) intracellular loop, YxxL and TxxS/xS motifs in the 3(rd) intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the 3(rd) intracellular loop markedly decreased the level of D(3)R expression compared with D(2)R. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between D(2)R and D(3)R in the 1(st) intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between D(2)R and D(3)R. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the 1(st) loop of D(3)R are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D(2)R has a more flexible overall conformation that can accept mutated residues in the intracellular region than D(3)R, which might be partly responsible for the quantitative differences in the signaling efficiency between D(2)R and D(3)R.     

Requirement of Gbetagamma and c-Src in D2 dopamine receptor-mediated nuclear factor-kappaB activation

The D2 dopamine receptor (D2R) was examined for its ability to mediate nuclear factor-kappaB (NF-kappaB) activation through G proteins. Stimulation of D2R-transfected HeLa cells with its agonist quinpirole induced the expression of a NF-kappaB luciferase reporter and formation of NF-kappaB-DNA complex. This response was blocked by pertussis toxin, and by the Gbetagamma scavengers transducin and beta-adrenergic receptor kinase 1 carboxyl-terminal fragment. Unlike Gi-coupled chemoattractant receptors, D2R activated NF-kappaB without an increase in phospholipase C-beta activity, and the response was only slightly affected by the phosphoinositide 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). In contrast, treatment with genistein and 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d] pyrimidine abolished the induced NF-kappaB activation, suggesting involvement of protein tyrosine kinases. Activation of D2R led to phosphorylation of c-Src at Tyr-418, and expression of a kinase-deficient c-Src inhibited D2R-mediated NF-kappaB activation. The D2R-mediated NF-kappaB activation was not dependent on epidermal growth factor (EGF) receptor transactivation since 4-(3'-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an EGF receptor-selective tyrphostin used at 1 microM, blocked EGF-induced NF-kappaB activation but not the quinpirole-induced response. In addition, the D2R-mediated NF-kappaB activation was enhanced by over-expression of beta-arrestin 1. These results suggest that D2R-mediated NF-kappaB activation requires Gbetagamma and c-Src, and possibly involves beta-arrestin 1.     

N-linked glycosylation is required for plasma membrane localization of D5, but not D1, dopamine receptors in transfected mammalian cells.

We have analyzed the role of N-linked glycosylation in functional cell surface expression of the D1 and D5 dopamine receptor subtypes. Treatment of transfected HEK 293 cells with tunicamycin, an inhibitor of N-linked oligosaccharide addition, was found to prevent localization of D5 receptors in the plasma membrane. In contrast, tunicamycin treatment had no effect on the plasma membrane localization of the D1 receptor. Polymerase chain reaction mutagenesis was used to generate a panel of D5 receptors containing mutations in the three predicted sites of N-linked glycosylation. Expression of mutant receptors indicated that glycosylation of residue N7 was the major determinant of D5 receptor plasma membrane localization. Mutation of a comparable site in the D1 receptor at position N5 had no effect on the delivery of the D1 receptor to the cell surface. Tunicamycin treatment during receptor biosynthesis, but not N-glycosidase F digestion of mature receptors, abrogated binding of the D5 receptor antagonist [(3)H]SCH23390, suggesting that while oligosaccharide moieties play a key role in the cell surface expression of D5 receptors, they do not appear to contribute to the receptor's ligand binding properties. Together, our data indicate a differential requirement for N-linked glycosylation in functional cell surface expression of D1 and D5 dopamine receptors.     

Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280)

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.     

Protein-protein interactions and dopamine D2 receptor signaling: a calcium connection

The third cytoplasmic loop is a crucial site of physical contact between some G protein-coupled receptors (GPCRs) and their respective G proteins. However, interactions not only occur among these proteins but also involve a number of additional protein binding partners. Modulation of these protein-protein interactions may represent an important new avenue of pharmacotherapy through which signaling of GPCRs can be modulated. In the current issue of Molecular Pharmacology, Liu et al. (p. 371) report that dopamine D(2) receptors interact with the Ca(2+) binding protein S100B. Using the third intracellular loop of the dopamine D(2) receptor as bait in a bacterial two-hybrid system, S100B was determined to be a potential binding partner. They used pull-down assays both in vitro and in vivo to confirm the interaction and define its specificity. Neither the D(3) nor the D(4) receptor expresses the motif conferring the interaction, and peptides based on the third intracellular loop of the D(3) receptor did not pull down S100B. Some groups might stop there, but Liu and colleagues moved on to demonstrate colocalization of the D(2) receptor and S100B by immunostaining. Functional assays were then used to show that coexpression of S100B with the D(2) receptor increases the ability of D(2) receptors to activate ERK and to inhibit adenylyl cyclase. These data suggest that S100B coexpression may serve as an important mediator of D(2) receptor signaling efficacy in the brain. These interactions contribute to cellular, regional, and developmental differences in D(2) receptor activation.     

The dopamine D3 receptor interacts with itself and the truncated D3 splice variant d3nf: D3-D3nf interaction causes mislocalization of D3 receptors

We have generated a stable cell line expressing FLAG epitope-tagged D3 dopamine receptors and used this cell line to study D3 receptor-protein interactions. To analyze protein interactions, we separately introduced into the stable cell line either D3 receptors carrying an hemagglutinin (HA) epitope tag, or an HA-tagged version of the D3 receptor splice variant D3nf. A combination of confocal laser microscopy and coimmunoprecipitation was used to assay the formation and expression pattern of D3-D3 homodimers or D3-D3nf heterodimers. When coexpressed in HEK 293 cells, FLAG- and HA-tagged D3 receptors exhibited a similar plasma membrane distribution. Using an HA epitope tag-specific antibody, we coimmunoprecipitated HA- and FLAG-tagged D3 receptors, suggesting that D3 receptors are capable of forming homodimers. Epitope-tagged D3nf polypeptides exhibited a markedly different cellular distribution than D3 receptors. When expressed in HEK 293 cells, either alone or in combination with FLAG-tagged D3 receptors, D3nf exhibited a punctate perinuclear distribution. When D3nf was introduced into the stable D3-expressing cell line, D3 receptors were no longer visualized at the plasma membrane. Instead, D3 and D3nf showed a similar, predominantly cytosolic, localization. Using the HA-specific antibody, we were able to coimmunoprecipitate D3 and D3nf polypeptides from transfected cells. These data suggest the existence of physical interaction between D3 and D3nf. This interaction appears to result in the mislocalization of D3 receptors from the plasma membrane to an intracellular compartment, a finding that could be of significance in the etiology of schizophrenia.     

Regulation of dense core vesicle release from PC12 cells by interaction between the D2 dopamine receptor and calcium-dependent activator protein for secretion (CAPS)

We identified CAPS1 (calcium-dependent activator protein for secretion) as a D2 dopamine receptor interacting protein (DRIP) in a yeast two-hybrid screen of a human brain library using the second intracellular domain of the human D2 receptor (D2IC2). CAPS1 is an evolutionarily conserved calcium binding protein essential for late-stage exocytosis of neurotransmitters from synaptic terminals. CAPS1 interaction was confirmed for both the long and short isoforms of the D2 receptor, but not with any other dopamine receptor subtype. Interaction between CAPS1 and the D2 receptor was validated using both pulldown and coimmunoprecipitation assays. Deletion mapping localized the D2 receptor binding site to a segment located within the C-terminal region of CAPS1 as well CAPS2. In PC12 cells, CAPS1 and D2 receptors were found to colocalize within both cytosolic and plasma membrane compartments. Overexpression of a truncated D2 receptor fragment caused a significant decrease in K(+)-evoked dopamine release from PC12 cells, whereas no effect on norepinephrine or BDNF release was observed. These results suggest that D2 dopamine receptors may modulate vesicle release from neuroendocrine cells via direct interaction with components of the exocytotic machinery.     

Characterisation of Gs activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine

Herein, we examined the direct coupling of human dopamine D1 receptors to G(s) proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists ( approximately 100%) in stimulating guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding to G(s) in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [35S]GTP gamma S binding to G(s) with a pK(B) (9.29) close to its pK(i) (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated G(s) activation with pK(B)'s of 6.7 and 7.3, respectively, values close to their pK(i) values at these sites. In conclusion, the use of an anti-G(s) protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.     

The influence of G protein subtype on agonist action at D2 dopamine receptors

In previous studies, we have shown that agonists influence the ability of D2 dopamine receptors to couple to G proteins and here we extend this work. The human D2Short dopamine receptor and a natural polymorphism of this D(2Short)(Ser311Cys), have been studied by co-expressing the receptors in insect cells with Gbeta1gamma2 and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[3H]spiperone competition binding experiments and through stimulation of [35S]GTPgammaS binding. Although the Ser311Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D2 receptor, agonist stimulation of [35S]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins.     

Mu-opioid receptor heterooligomer formation with the dopamine D1 receptor as directly visualized in living cells

Our immunohistochemistry experiments demonstrated that the mu-opioid receptor co-localized with the dopamine D1 receptor in neurons of the cortex and caudate nucleus. On the basis of this physiological data we further investigated whether these two G protein coupled receptors formed hetero-oligomers in living cells. To demonstrate hetero-oligomerization we used a novel strategy, the method used harnessed the physiological cellular mechanism for transport of proteins to the nucleus. The nuclear translocation pathway was adapted for the visualization of mu-opioid hetero-oligomers with the dopamine D1 receptor. The receptor hetero-oligomer complex formed resulted in a significantly enhanced surface expression of mu-opioid receptor. This hetero-oligomer formation involved the interaction of mu-opioid receptor with the dopamine D1 receptor carboxyl tail, since a dopamine D1 receptor substituted with the carboxyl of the dopamine D5 receptor failed to increase surface expression of mu-opioid receptor.     

Adenosine A2A receptor and dopamine D3 receptor interactions: evidence of functional A2A/D3 heteromeric complexes

Adenosine A(2A) and dopamine D(2) receptors have been shown previously to form heteromeric complexes and interact at the level of agonist binding, G protein coupling, and trafficking. Because dopamine D(2) and D(3) receptors show a high degree of sequence homology, A(2A) and D(3) receptors may also interact in a similar manner. The present studies with confocal microscopy showed that A(2A)-yellow fluorescent protein (YFP) and D(3)-green fluorescent protein 2 (GFP2) receptors colocalize in the plasma membrane. Furthermore, fluorescence resonance energy transfer (FRET) analysis demonstrated that A(2A)-YFP and D(3)-GFP2 receptors give a positive FRET efficiency and are thereby likely to exist as heteromeric A(2A)/D(3) receptor complexes. Saturation experiments with [(3)H]dopamine demonstrated that the A(2A) receptor agonist 4-[2-[[6-amino-9(N-ethyl-beta-d-ribofuranuronaminoamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS-21680) reduced the affinity of the high-affinity agonist binding state of the D(3) receptor for [(3)H]dopamine. The A(2A) and D(2A) receptors seem to interact also at the level of G protein coupling, because the adenosine A(2A) receptor agonist CGS-21680 fully counteracted the D(3) receptor-mediated inhibition of a forskolin-mediated increase in cAMP levels. Taken together, when coexpressed in the same neuron, A(2A) and D(3) receptors seem to form A(2A)/D(3) heteromeric receptor complexes in which A(2A) receptors antagonistically modulate both the affinity and the signaling of the D(3) receptors. D(3) receptor is one of the therapeutic targets for treatment of schizophrenia, and therefore, the A(2A)/D(3) receptor interactions could provide an alternative antischizophrenic treatment.     

A proteomic approach to receptor signaling: molecular mechanisms and therapeutic implications derived from discovery of the dopamine D2 receptor signalplex

Recent research in cell signaling has shown that the assembly of G protein coupled receptors into signaling complexes or signalplexes represents the primary mechanism by which receptor-mediated signaling is established and maintained. In this review, we summarize the current state of knowledge regarding protein interactions that comprise the dopamine D2 receptor signalplex within the brain. Studies based on conventional and advanced two-hybrid methodologies, as well as bioinformatic and computational analysis of sequence information from completed genomes have demonstrated interactions between dopamine D2 receptors and a cohort of dopamine receptor interacting proteins (DRIPs). DRIP interactions appear to regulate key aspects of receptor function including the signaling and membrane trafficking of dopamine D2 receptors. Disruptions or modifications of the signalplex, using membrane permeant competing peptide or dominant negative approaches, may represent promising new strategies for the selective targeting of the dopamine D2 receptor in cells and in native tissue. DRIP interactions provide a novel platform for understanding the mechanisms of dopamine receptor signaling, and for the potential development of novel treatments for brain disease.     

Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors enhances heterodimer formation: a mechanism for receptor cross-talk?

Dopamine and endogenous cannabinoids display complex interactions in the basal ganglia. One possible level of interaction is between CB1 cannabinoid and D2 dopamine receptors. Here, we demonstrate that a regulated association of CB1 and D2 receptors profoundly alters CB1 signaling. This provides the first evidence that CB1/D2 receptor complexes exist, are dynamic, and are agonist-regulated with highest complex levels detected when both receptors are stimulated with subsaturating concentrations of agonist. The consequence of this interaction is a differential preference for signaling through a "nonpreferred" G protein. In this case, D2 receptor activation, simultaneously with CB1 receptor stimulation, results in the receptor complex coupling to G alpha s protein in preference to the expected G alpha i/o proteins. The result of this interaction is an increase in the second messenger cAMP, reversing an initial synergistic inhibition of adenylyl cyclase activity seen at subthreshold concentrations of cannabinoid agonist. Additionally, a pertussis toxin insensitive component in the activation of extracellular signal-regulated kinase (ERK) 1/2 kinases by the cannabinoid agonist CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] is revealed in cells stably expressing both CB1 and D2 receptors. Thus, concurrent receptor stimulation promotes a heterooligomeric receptor complex and an apparent shift of CB1 signaling from a pertussis toxin-sensitive inhibition to a partly pertussis toxin-insensitive stimulation of adenylyl cyclase and ERK 1/2 phosphorylation.     

D(4) dopamine receptor differentially regulates Akt/nuclear factor-kappa b and extracellular signal-regulated kinase pathways in D(4)MN9D cells

The present study was designed to investigate the role of D(4) dopamine receptors in regulating the Akt/nuclear factor-kappa B (NF-kappa B) and extracellular signal-regulated kinase (ERK) signaling pathways. The D(4) dopamine receptor agonist PD168077 induced time- and dose-dependent activation of Akt and ERK in D(4)MN9D cells that stably express D(4) dopamine receptors. Maximal Akt and ERK stimulation was achieved at 1 microM PD168077. The agonist-mediated stimulations of Akt and ERK were abolished when cells were preincubated with 50 ng/ml PTX or with 1 microM L745,870, a D(4) dopamine receptor antagonist, indicating that activation of the Akt or ERK pathways is mediated by D(4) dopamine receptors and require a pertussis toxin-sensitive G protein. We also detected a time- and dose-dependent activation of NF-kappa B. Activation of NF-kappa B by 1 microM PD168077 was attenuated in D(4)MN9D cells that were transfected with a kinase-deficient Akt but not in cells transfected with a dominant negative Ras (N17Ras), suggesting that NF-kappa B activation requires Akt but is independent of Ras. In contrast, the transfection of N17Ras into D(4)MN9D cells blunted D(4) dopamine receptor-mediated ERK activation, indicating a Ras-dependent mechanism. Moreover, PP2 (20 nM), an inhibitor of Src, blocked D(4) receptor-mediated SHC phosphorylation and ERK activation. In contrast, transfection of a kinase-dead Akt did not alter D(4) receptor-stimulated ERK. However, PP2 and the mitogen activated protein kinase kinase inhibitor PD98059 did not change D(4) receptor-mediated Akt/NF-kappa B activation. All these indicate that distinct mechanisms mediate ERK and Akt/NF-kappa B activation by D(4) dopamine receptor stimulation. We also demonstrated that D(4) receptor-stimulated cell proliferation is mediated by the Src/SHC/Ras/ERK pathway.     

Functional coupling of the human dopamine D2 receptor with G alpha i1, G alpha i2, G alpha i3 and G alpha o G proteins: evidence for agonist regulation of G protein selectivity

(1) The human dopamine D(2long) (D(2L)) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)), the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 micro M), demonstrating interaction between the receptor and the different G proteins. (2) The receptor to G protein ratio (R : G ratio) was evaluated using [(3)H]-spiperone saturation binding (R) and [(35)S]-GTPgammaS saturation binding (G). R : G ratios of 1 : 12, 1 : 3, 1 : 14 and 1 : 5 were found for G(i1), G(i2), G(i3), and G(o) preparations, respectively. However, when R : G ratios of 1 : 2 and 1 : 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [(35)S]-GTPgammaS binding. (3) Several agonists were tested for their ability to stimulate [(35)S]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [(35)S]-GTPgammaS binding. (4) Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. (5) We conclude that the degree of selectivity of G protein activation by the D(2L) receptor can depend on the conformation of the receptor stabilised by an agonist.     

Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors

The recently cloned protein, calcyon, potentiates crosstalk between G(s)-coupled dopamine D1 receptors and heterologous G(q/11)-coupled receptors allowing dopamine D1 receptors to stimulate intracellular Ca(2+) release, in addition to cAMP production. This crosstalk also requires the participating G(q/11)-coupled receptors to be primed by their agonists. We examined the ability of calcyon and priming to regulate the affinity of dopamine D1 receptors for its ligands. Receptor binding assays were performed on HEK293 cell membrane preparations expressing dopamine D1 receptors either alone or in combination with calcyon. Co-expression of dopamine D1 receptor and calcyon affected neither the affinity of this receptor for antagonists nor the affinity of agonist binding to this receptor high and low-affinity states. However, the presence of calcyon dramatically decreased the proportion of the high-affinity dopamine D1 receptor agonist binding sites. This decrease was reversed by carbachol, which primes the receptor crosstalk by stimulating endogenous G(q/11)-coupled muscarinic receptors. Our findings suggest that calcyon regulates the ability of dopamine D1 receptors to achieve the high-affinity state for agonists, in a manner that depends on priming of receptor crosstalk.     

Biochemical and cell biological mechanisms of cholecystokinin receptor regulation

Regulation of the cholecystokinin receptor is accomplished by biochemical and cell biological mechanisms. The major mechanism for biochemical regulation involves phosphorylation of serine and threonine residues within the receptor's intracellular third loop and carboxyl-terminal tail. This form of rapid desensitization is achieved by protein kinase C, a kinase activated in the normal signaling cascade of this Gq-coupled receptor, and/or a member of the G protein-coupled receptor kinase family that recognizes the active conformation of the receptor. Conversely, a type 2A serine protein phosphatase has been shown to selectively act on this receptor in an agonist-regulated manner, resulting in receptor dephosphorylation and resensitization. Cell biological mechanisms for desensitization involve the net removal of receptors from exposure to circulating hormone via insulation within a specialized plasma membrane domain or internalization into the cell within endocytic compartments. Internalization has been shown to occur by both clathrin-dependent and clathrin-independent mechanisms, the latter believed to represent potocytosis in caveolae, that lead to recycling of receptors to the plasma membrane for resensitization or shuttling of receptors to lysosomes for degratory down-regulation. Interestingly, receptor phosphorylation has been shown to affect intracellular domain accessibility and receptor trafficking, although the specific proteins regulating this have not yet been identified. The cholecystokinin receptor can exist in the plasma membrane as oligomeric superstructures, namely as homomers with themselves or as heteromers with closely related class I G protein-coupled receptors. Agonist occupation results in oligomer dissociation, but the functional significance of this observation has yet to be defined.