Signaling Mechanisms of the D3 Dopamine Receptor

A substantial body of evidence shows the capacity of the dopamine D₃ receptor to couple functionally to G proteins when expressed in an appropriate milieu in heterologous expression systems. In these systems, activation of D₃ receptors inhibits adenylate cyclase, modulates ion flow through potassium and calcium channels, and activates kinases, most notably mitogen-activated protein kinase. Coupling to G_i/G_o is implicated in many of these effects, but other G proteins may contribute. Studies with chimeric receptors implicate the third intracellular loop in the mediation of agonist-induced signal transduction. Finally, D₃-preferring drugs modulate expression of c-fos in neuronal cultures and brain. Signaling mechanisms of the D₃ receptor in brain, however, remain to be definitively determined.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

β-arrestin2 plays permissive roles in the inhibitory activities of RGS9-2 on G protein-coupled receptors by maintaining RGS9-2 in the open conformation

Together with G protein-coupled receptor (GPCR) kinases (GRKs) and β-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D(3) receptor (D(3)R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, β-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, Gβ5, R7-binding protein (R7BP), and D(3)R. The DEP domain of RGS9-2, under the permission of β-arrestin2, inhibited the signaling of D(3)R in collaboration with Gβ5. β-Arrestin2 competed with R7BP and Gβ5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for β-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that β-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.     

Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors.

Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor.     

Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system

The selectivity of D2 dopamine receptor-guanine nucleotide-binding protein (G protein) coupling was studied by reconstitution techniques utilizing purified D2 dopamine receptors from bovine anterior pituitary and resolved G proteins from bovine brain, bovine pituitary, and human erythrocyte. Titration of a fixed receptor concentration with varying G protein concentrations revealed two aspects of receptor-G protein coupling. First, Gi2 appeared to couple selectively with the D2 receptor with approximately 10-fold higher affinity than any other tested Gi subtype. Second, the G proteins differed in the maximal receptor-mediated agonist stimulation of the intrinsic GTPase activity. Gi2 appeared to be maximally stimulated by agonist-receptor complex with turnover numbers of approximately 2 min-1. The other Gi subtypes, Gi1 and Gi3, could be only partially activated, resulting in maximal rates of GTPase of approximately 1 min-1. Agonist-stimulated GTPase activity was not detected in preparations containing Go from bovine brain. The differences in maximal agonist-stimulated GTPase rates observed among the Gi subtypes could be explained by differences in agonist-promoted guanyl nucleotide exchange. Both guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding and GDP release parameters were enhanced 2-fold for the Gi2 subtype over the other Gi subtypes. These results suggest that even though several types of pertussis toxin substrate may exist in most tissues, a receptor may interact discretely with G proteins, thereby dictating signal transduction mechanisms.     

Distinct roles for Galphai2, Galphai3, and Gbeta gamma in modulation offorskolin- or Gs-mediated cAMP accumulation and calcium mobilization by dopamine D2S receptors

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.     

Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts

The 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1 and the expression of bone matrix proteins. Reports differ on the effects of IGF binding protein (IGFBP)-5 on bone cell growth and osteoblastic function. IGFBP-5 can be growth stimulatory or inhibitory and can enhance or impair osteoblast function. In previous studies, we have shown that IGFBP-5 localizes to the nucleus and interacts with the retinoid receptors. We now show that IGFBP-5 interacts with nuclear vitamin D receptor (VDR) and blocks retinoid X receptor (RXR):VDR heterodimerization. VDR and IGFBP-5 were shown to colocalize to the nuclei of MG-63 and U2-OS cells and coimmunoprecipitate in nuclear extracts from these cells. Induction of osteocalcin promoter activity and alkaline phosphatase activity by 1,25(OH)2D3 were significantly enhanced when IGFBP-5 was down-regulated in U2-OS cells. Moreover, we found IGFBP-5 increased basal alkaline phosphatase activity and collagen alpha1 type 1 expression, and that 1,25(OH)2D3 was unable to further induce the expression of these bone differentiation markers in MG-63 cells. Expression of IGFBP-5 inhibited MG-63 cell growth and caused cell cycle arrest at G0/G1 and G2/M. Furthermore, IGFBP-5 reduced the effects of 1,25(OH)2D3 in blocking cell cycle progression at G0/G1 and decreased the expression of cyclin D1. These results demonstrate that IGFBP-5 can interact with VDR to prevent RXR:VDR heterodimerization and suggest that IGFBP-5 may attenuate the 1,25(OH)2D3-induced expression of bone differentiation markers while having a modest effect on the 1,25(OH)2D3-mediated inhibition of cell cycle progression in bone cells.     

The dopamine D4 receptor,the ultimate disordered protein

The human D4 dopamine receptor is a synaptic neurotransmitter receptor responsible for neuronal signaling in the mesolimbic system of the brain, an area of the brain that regulates emotion and complex behavior. Its structure makes it a very unusual and interesting G protein-coupled receptor (GPCR) as it has several polymorphic variants of its gene in the region encoding the third intracellular loop (IL3). This region contains from two to seven or more similar 48 base pair repeats. These repeats cause this protein to have a very high disorder index and this, in turn, makes it very interactive with other proteins. Among GPCRs in general, the unusually proline-rich IL3 is unique to the D4 receptor (D4R). We believe that, as in the D2R, this region of the receptor plays a role in it’s interaction with other receptors.     

Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse

Protein therapeutics may be delivered across the blood-brain barrier (BBB) by genetic fusion to a BBB molecular Trojan horse. The latter is an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) against a BBB receptor, such as the insulin receptor or the transferrin receptor (TfR). Fusion proteins have been engineered with the MAb against the human insulin receptor (HIR). However, the HIRMAb is not active against the rodent insulin receptor, and cannot be used for drug delivery across the mouse BBB. The rat 8D3 MAb against the mouse TfR is active as a drug delivery system in the mouse, and the present studies describe the cloning and sequencing of the variable region of the heavy chain (VH) and light chain (VL) of the rat 8D3 TfRMAb. The VH and VL were fused to the constant region of mouse IgG1 heavy chain and mouse kappa light chain, respectively, to produce a new chimeric TfRMAb. The chimeric TfRMAb was expressed in COS cells following dual transfection with the heavy and light chain expression plasmids, and was purified by protein G affinity chromatography. The affinity of the chimeric TfRMAb for the murine TfR was equal to the 8D3 MAb using a radio-receptor assay and mouse fibroblasts. The chimeric TfRMAb was radio-labeled and injected into mice for a pharmacokinetics study of the clearance of the chimeric TfRMAb. The chimeric TfRMAb was rapidly taken up by mouse brain in vivo at a level comparable to the rat 8D3 MAb. In summary, these studies describe the genetic engineering, expression, and validation of a chimeric TfRMAb with high activity for the mouse TfR, which can be used in future engineering of therapeutic fusion proteins for BBB drug delivery in the mouse.     

Influence of the antipsychotic drug pipamperone on the expression of the dopamine D4 receptor

The dopamine D4 receptor is a G protein-coupled receptor that binds with high affinity various antipsychotics. The receptor may be involved in attention/cognition, and in genetic studies a polymorphic repeat sequence in its coding sequence has been associated with attention deficit/hyperactivity disorder. We developed an inducible episomal expression system based on the reverse tetracycline transactivator and Epstein-Barr viral sequences. In HEK293rtTA cells expressing the dopamine D4 receptor from this episomal expression vector, addition of doxycycline in combination with sodium butyrate and trichostatin A induces high levels of receptor expression, resulting in 1970 +/- 20 fmol/mg membrane protein. Addition of the dopamine D4 receptor and serotonin 5-HT2A receptor antagonist pipamperone to these cells further increased the expression of the dopamine receptor, reaching 3800 +/- 60 fmol/mg membrane protein. This up-regulation was not restricted to the dopamine D4 receptor but was also found for the serotonin 5-HT2A receptor. We further provide evidence that the increase in receptor expression is not due to increased mRNA synthesis. As pipamperone could rescue the expression of a folding mutant of the dopamine D4 receptor (M345), we propose that pipamperone acts as a pharmacological chaperone for correct receptor folding thereby resulting in an increased dopamine D4 receptor expression. This study describes a strong and inducible expression system for proteins, difficult to express in other heterologous expression systems. This study also demonstrates that pipamperone, an antipsychotic, acts as a pharmacological chaperone and by doing so, increases the expression level of the dopamine D4 receptor. The fact that ligands can also act as pharmacological chaperones is a fairly new additional element in the regulation of receptor expression levels with potential great impact in drug treatment.     

G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins.     

Evidence that calmodulin binding to the dopamine D2 receptor enhances receptor signaling

The Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D2 receptor. We confirmed that the third intracellular loop of the D2 receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D2-like receptor agonist 7-OH-DPAT increased the colocalization of the D2 receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D2-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D2 receptor and CaM and also significantly decreased D2 receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D2 receptor enhances D2 receptor signaling.     

CLIC4 interacts with histamine H3 receptor and enhances the receptor cell surface expression

Histamine H3 receptor (H3R), one of G protein-coupled receptors (GPCRs), has been known to regulate neurotransmitter release negatively in central and peripheral nervous systems. Recently, a variety of intracellular proteins have been identified to interact with carboxy (C)-termini of GPCRs, and control their intracellular trafficking and signal transduction efficiencies. Screening for such proteins that interact with the C-terminus of H3R resulted in identification of one of the chloride intracellular channel (CLIC) proteins, CLIC4. The association of CLIC4 with H3R was confirmed in in vitro pull-down assays, coimmunoprecipitation from rat brain lysate, and immunofluorescence microscopy of rat cerebellar neurons. The data from flowcytometric analysis, radioligand receptor binding assay, and cell-based ELISA indicated that CLIC4 enhanced cell surface expression of wild-type H3R, but not a mutant form of the receptor that failed to interact with CLIC4. These results indicate that, by binding to the C-terminus of H3R, CLIC4 plays a critical role in regulation of the receptor cell surface expression.     

An aspartate residue at the extracellular boundary of TMII and an arginine residue in TMVII of the gastrin-releasing peptide receptor interact to facilitate heterotrimeric G protein coupling.

The mammalian bombesin receptor subfamily of G protein-coupled receptors currently consists of the gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor, and bombesin receptor subtype 3. All three receptors contain a conserved aspartate residue (D98) at the extracellular boundary of transmembrane domain II and a conserved arginine residue (R309) near the extracellular boundary of transmembrane domain VII. To evaluate the functional role of these residues, site-directed GRP-R mutants were expressed in fibroblasts and assayed for their ability to both bind agonist and catalyze exchange of guanine nucleotides. Alanine substitution at GRP-R position 98 or 309 reduced agonist binding affinity by 24- and 56-fold, respectively, compared to wild-type GRP-R. Single swap GRP-R mutations either resulted in no receptor expression in the membrane (D98R) or the protein was not able to bind agonist (R309D). In contrast, the double swap mutation (D98R/R309D) had high-affinity agonist binding, reduced from wild-type GRP-R by only 6-fold. In situ reconstitution of urea-extracted membranes expressing either wild-type or mutant (D98A or R309A) GRP-R with G(q) indicated that alanine substitution greatly reduced G protein catalytic exchange compared to wild-type GRP-R. The D98R/R309D GRP-R had both a higher intrinsic basal activity and a higher overall catalytic exchange activity compared to wild-type; however, the wild-type GRP-R produced a larger agonist-stimulated response relative to the double swap mutant. Taken together, these data show that GRP-R residues D98 and R309 are critical for efficient coupling of GRP-R to G(q). Furthermore, our findings are consistent with a salt bridge interaction between these two polar and oppositely charged amino acids that maintains the proper receptor conformation necessary to interact with G proteins.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.     

Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.