Dopamine Receptor Oligomerization Visualized in Living Cells

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist SKF38393, we found that Trk neurotrophin receptors were activated in embryonic day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by SKF38393 was accompanied by the phosphorylation of several Trk signaling proteins, including phospholipase Cgamma, Akt, and MAPK. Biotinylation experiments followed by immunostaining by phospho-TrkB-specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca(2+). These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate the effects of dopamine upon striatal neurons.

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D(2) dopamine receptor (DAR). Agonist activation of D(2) DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D(2) DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [(35)S]GTPgammaS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D(2) DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.

Striatal Plasticity and Basal Ganglia Circuit Function

REVIEW ARTICLES: Platelet G protein-coupled receptors in hemostasis and thrombosis

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical alpha2A-adrenergic receptor (alpha2A AR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether alpha(2)AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the alpha2A AR (Val(217)-Ala(377)), alpha2BAR (Lys(210)-Trp(354)), and alpha2CAR (Arg(248)-Val(363)) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because alpha2A AR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to alpha2 AR localization but also to agonist modulation of alpha2AR signaling.

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.