The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.

RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

Influence of benzodiazepine binding site ligands on fear-conditioned contextual memory.

Eight compounds that bind to the benzodiazepine binding site on the gamma-amino butyric acid(A) (GABA(A)) receptor were assessed for their influence on contextual memory, an aspect of memory affected in various cognitive disorders including Alzheimer's disease. Using a Pavlovian fear-conditioning paradigm, each ligand was evaluated in C57Bl/6 mice in regards to its direct affect on contextual memory and whether the ligand could attenuate scopolamine-induced contextual memory impairment. Of the eight ligands tested, one impaired contextual memory (agonist), six attenuated scopolamine-induced contextual memory impairment (inverse agonists), and one antagonized the ability of an inverse agonist to attenuate scopolamine-induced contextual memory impairment. Hence, further demonstrating the bi-directional influence benzodiazepine binding site ligands are able to exert on memory modulation. This study serves as an initial starting point in the development of pharmacological tools to be used in deciphering how GABA(A) receptors influence contextual memory.

Characterization of novel benzodiazepine ligands in Spodotera frugiperda (Sf-9) insect cells.

The goals of the present work were to characterize the binding profile of nine benzodiazepine ligands in Spodotera frugiperda (Sf-9) insect cells expressing specific gamma aminobutyric acid (A) (GABA(A)) receptor subunit combinations and compare the affinities to those for the receptors in the rat cerebellum. Three recombinant baculovirus constructs, each harboring a different GABA(A) receptor subunit, were introduced into insect cells by simultaneous infection. Saturation and competition binding assays were carried out in membranes from Sf-9 cells infected with either alpha1beta2gamma2 or alpha6beta2gamma2 subunit combinations. The affinities of the ligands to the alpha1beta2gamma2 or alpha6beta2gamma2 receptors expressed in Sf-9 cells were similar to the affinities previously determined for the alpha1 or alpha6 subunit-containing GABA(A) receptors in the rat cerebellum, respectively, thus confirming the previously assigned receptor types in the cerebellum.

Characterization of benzodiazepine receptors in the cerebellum.

1. The goals of the work reported here were to further characterize benzodiazepine/GABA(A) (BDZR) receptor heterogeneity in the cerebellum and to measure the affinities and selectivities of structurally diverse benzodiazepines at each site identified. 2. Five chemical families were included in these studies. These were 1,4-benzodiazepines (flunitrazepam), imidazobenzodiazepines (RO15-1788 and RO15-4513 and RO16-6028), beta-carbolines (Abecarnil) and pyrazoloquinolines (CGS 8216, CGS 9895 and CGS 9896). 3. Saturation and competition binding assays were combined with powerful data analysis software developed in our laboratory. Among the capabilities of this software is the identification of multiple binding sites for a cold ligand using a non-selective labeled ligand that binds with equal, but high, affinity to all the binding sites 4. Saturation binding assays using either [3H]-RO15-1788 or [3H]-RO15-4513 revealed only one apparent binding site, with a higher affinity for RO15-4513 than for RO15-1788. However, using [3H]-RO15-4513 for the competition binding studies in the cerebellum, together with our data analysis software, led to the identification of two distinct binding sites with equal densities for the diverse benzodiazepines studied. 5. In rat cerebellum one of the sites identified corresponds to GABA(A) receptors exhibiting alpha1 subunit pharmacology and the other to GABA(A) receptors exhibiting alpha6 subunit pharmacology. In general, the diverse families of BDZR ligands studied had much lower affinities for the alpha6 containing receptors.

Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb.

The goals of the work reported here were (i) to identify distinct GABA(A)/benzodiazepine receptors in the rat hippocampus and olfactory bulb using receptor binding assays, and (ii) to determine the affinities and selectivities of benzodiazepine receptor ligands from structurally diverse chemical families at each site identified. These studies were aided by the use of software AFFINITY ANALYSIS SYSTEM, developed in our laboratory for analysis of receptor binding data that allows the determination of receptor heterogeneity using non-selective radioligands. Saturation binding assays using [3H]RO15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a]-[1,4]benzodiazepine-3-carboxylate) revealed two binding sites in each of these two tissues. The higher affinity site corresponds to alpha(5) subunit-containing GABA(A) receptor and the lower affinity site to a combination of alpha(1), alpha(2), and alpha(3) subunit-containing receptors. These results should be useful in the challenging task of identifying the various functional GABA(A) receptors in the central nervous system, and in providing a link between receptor affinities and in vivo activities of the GABA(A)/benzodiazepine receptor ligands studied.

Differential regulation of muscarinic M1 and M3 receptors by a putative phosphorylation domain.

A motif consisting of several serine residues flanked N-terminally by acidic residues occurs in the third intracellular loop of both muscarinic M1 and M3 receptors (287SerLeuThrSerSer291 and 349SerAlaSerSer352, respectively). We examined the role of these domains in modulating agonist-induced desensitization and receptor trafficking, and for the muscarinic M3 receptor, we assessed the contribution of phosphorylation to receptor regulation. Mutation of the above residues did not affect desensitization of phosphoinositide hydrolysis signaling for either the muscarinic M1 or M3 receptor and did not alter the agonist-induced phosphorylation state of the muscarinic M3 receptor. Mutation of this domain (349SerAlaSerSer352/349AlaAlaAlaAla352) in the muscarinic M3 receptor completely abrogated receptor internalization and subsequently, down-regulation. Mutation of the analogous domain (287SerLeuThrSerSer291/287AlaLeuAlaAlaAla291) in the muscarinic M1 receptor had no obvious effect on internalization, but led to a more rapid down-regulation. Thus, these serine-rich regions are not required for receptor desensitization, but are differentially involved in receptor trafficking for the muscarinic M1 and M3 receptors.

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.

Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein.

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.

Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

Antibody to epitope tag induces internalization of human muscarinic subtype 1 receptor.

We have studied the effect of an antibody against the epitope EYMPME on the internalization of the human muscarinic cholinergic receptor hm1 tagged with the epitope at the amino terminus. The antibody to the tag induces internalization of the hm1 receptor within minutes after exposure of human embryonic kidney 293 cells transfected with the tagged receptor. This antibody-induced internalization is reversible following removal of the antibody. In contrast to hm1 internalization induced by the agonist carbachol, internalization induced by antibody is not blocked by the muscarinic antagonist atropine. The mechanism of antibody-mediated internalization does not appear to involve receptor dimerization by the antibody, as Fab fragments derived from the antibody also induce internalization. The pathway of antibody-induced internalization, similar to the agonist-induced process, is mediated by clathrin-coated vesicles. Furthermore, antibody treatment does not result in any second messenger production, as measured by phosphoinositide accumulation. Our data show that internalization of a G protein-coupled receptor can be triggered by interaction of the amino terminus of the receptor with an exogenous ligand and can occur independently of second messenger production. This result suggests that the receptor can exist in multiple conformations, each mediating distinct downstream events.

A constitutively internalizing and recycling mutant of the mu-opioid receptor.

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in mu-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C-terminus of the mu-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these mu-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wild-type and mutant receptors. Both the wild-type mu-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress mu-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.

Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.

Human muscarinic cholinergic receptor Hm1 internalizes via clathrin-coated vesicles.

The mechanism by which muscarinic receptors internalize upon agonist exposure is poorly understood. To determine the endocytic pathways responsible for muscarinic receptor internalization, we have stably transfected human embryonic kidney (HEK 293) cells with the Hm1 (human muscarinic subtype 1) receptor tagged at the amino terminus with the epitope EYMPME. The subcellular location of the receptor was visualized by immunofluorescence confocal microscopy and quantified with the use of binding studies. The receptor redistributed into intracellular compartments following agonist treatment. This process was reversible upon removal of agonist and inhibited by antagonist. Acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, inhibited carbachol-stimulated internalization. Phorbol 12-myristate 13-acetate, on the other hand, which inhibits caveolae-mediated endocytosis, had no effect on carbachol-induced endocytosis. Double-labeling confocal microscopy was used to characterize the intracellular vesicles containing Hm1 receptor following agonist treatment. The Hm1 receptor was shown to be colocalized with clathrin and alpha-adaptin, a subunit of the AP2 adaptor protein which links endocytosed proteins with clathrin in the intracellular vesicles. In addition, endosomes containing Hm1 also contained the transferrin receptor, which internalizes via clathrin-coated vesicles. In contrast, caveolin, the protein that comprises caveolae, did not colocalize with Hm1 in intracellular vesicles following agonist treatment, indicating that caveolae are not involved in the agonist-induced internalization of Hm1. These results indicate that agonist-induced internalization of the Hm1 receptor occurs via clathrin-coated vesicles in HEK cells.

Phosphorylation and agonist-specific intracellular trafficking of an epitope-tagged mu-opioid receptor expressed in HEK 293 cells.

We expressed the cloned mu-opioid receptor (muR) in high abundance (5.5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate] and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with [gamma-32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.

Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors.

The N- and C-terminal junctions of the third intracellular loop (i3) of G protein-coupled receptors play a role in the coupling process. We had previously constructed two triple point alanine mutants of the i3 junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K362A/T366A, which are defective in mediating carbachol stimulation of phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corresponding six single point mutations were constructed to determine residues crucial to receptor coupling. Mutants W209A and T366A were similar to or only slightly less effective than wild type Hm1 in stimulating PI turnover. In the N-terminal junction, I211A and Y212A were defective in coupling, and I211A was even more defective than the corresponding triple mutant. Therefore, the triple mutation compensated at least partially for the effect of these two single point mutations. In the C-terminal i3 loop junction, mutant K362A was again more strongly defective than the corresponding triple mutant. In contrast, mutation E360A was found to be activating, leading to elevated PI turnover in the absence of agonist and sensitization toward carbachol activation. Activating mutations in the C-terminal i3 loop junction have been reported previously for the adrenergic receptors, but E360A represents the first muscarinic receptor with substantial basal activity. The effects of the single point mutations observed in this study were not readily predictable from similar mutations from closely related G protein-coupled receptors despite sequence conservation in the i3 loop junctions. Our results caution against defining precise coupling domains in these regions by mutagenesis results.

Overlapping multi-site domains of the muscarinic cholinergic Hm1 receptor involved in signal transduction and sequestration.

Alanine mutagenesis scanning of the intracellular portion of the human muscarinic cholinergic Hm1 receptor was performed to identify domains mediating agonist induced receptor sequestration. Using these multiple alanine point mutants of Hm1, we had previously identified several receptor domains in the intracellular loops i1-3 that play a role in coupling to phosphatidyl inositol turnover, most notably, a lipophilic residue, Leu-131, in the conserved i2 loop domain DRYXXVXXPL (Moro, O., Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 6862-6865). We now demonstrate that alanine substitutions in three of these domains, i.e. middle of the i2 loop and both junctions of the i3 loop, also result in defective sequestration (loss of surface receptor sites accessible to a polar tracer) in transfected human kidney U293 cells. The i2 loop was studied further by single point mutations. The strongest impairment of sequestration occurred with mutant L131A which was also highly defective in phosphatidyl inositol (PI) coupling. Substitution of Leu-131 with several distinct amino acids indicated that a bulky lipophilic residue is required for sequestration in this position, as shown for coupling to PI turnover. Further, the double point mutation, V127A/L131A, almost completely suppressed both sequestration and coupling of Hm1. In the beta 2 adrenoceptor, alanine substitution of the i2 residue Phe-139, equivalent to Leu-131 in Hm1, also resulted in impaired coupling to adenylyl cyclase and sequestration, indicating a general role for this conserved i2 loop residue in both processes. The combined results show that the multi-site domain involved in signal transduction of Hm1 is similar to and overlaps with that involved in sequestration. However, three Hm1 mutants that were moderately deficient in stimulating PI turnover displayed normal sequestration, suggesting distinct mechanisms. We propose that cellular mediators of receptor sequestration are structurally similar or identical to the heterotrimeric G proteins.

Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.

Signal transduction of the heptahelical G protein-coupled receptors (GPCRs) involves multiple receptor domains, but a universal consensus domain for coupling has not yet been defined. Alanine mutagenesis scanning was performed on the intracellular loops and the COOH tail of the human muscarinic cholinergic receptor (Hm1) to identify coupling domains. Stimulation of phosphatidylinositol (PI) turnover was determined after transfection of the alanine mutants into U293 human embryonic kidney cells. Alanine substitutions in four regions (loops i1, i2, and NH2 and COOH junctions of i3) impaired coupling efficiency by approximately 50% or more, but the strongest reduction (> 80%) resulted from alanine replacement of a single amino acid, leucine 131. This residue is located in the middle of the second intracellular loop (i2), within the highly conserved GPCR motif (DRYXXV(I)XXPL). The position equivalent to Leu-131 in Hm1 contains a bulky hydrophobic amino acid (L, I, V, M, or F) in nearly all cloned GPCRs. Substitution of Leu-131 with polar amino acids (aspartate and asparagine) also resulted in strongly defective coupling, whereas phenylalanine (found in the equivalent position in the beta 2 adrenoceptor) can replace leucine without losing PI coupling ability of Hm1. Alanine substitution of the corresponding amino acid in the Hm3 receptor (L174A) also inhibited agonist-stimulated PI turnover, while replacing Phe-139 with alanine in the beta 2 adrenoceptor suppressed stimulation of adenylyl cyclase. We propose that a bulky hydrophobic amino acid in the middle of the i2 loop serves as a general site relevant to G protein coupling, whereas coupling selectivity is governed by other receptor domains.

Serine- and threonine-rich domain regulates internalization of muscarinic cholinergic receptors.

Upon agonist exposure, most membrane receptors internalize into the cell as part of an adaptation process. Receptor domains that mediate internalization have been defined for several receptor classes, e.g. growth factor and transport receptors, but not yet for any of the numerous members of the family of G protein-coupled receptors (GPCRs), having seven putative transmembrane helices. With the use of deletion mutations, we previously showed that a small region in the middle of the third intracellular loop (i3) appears to be required for the agonist-induced internalization of the Hm1 muscarinic cholinergic receptor (Lameh, J., Philip, M., Sharma, Y. K., Moro, O., Ramachandran, J., and Sadée, W. (1992) J. Biol. Chem. 267, 13406-13412). Using point mutations, we now demonstrate that domain 286-292 (ESLTSSE) is required, with TSS playing a crucial role. Although the i3 loops of GPCRs share minimal overall sequence identity, even among closely related subtypes, small domains containing multiple S/T residues are also present in several other GPCRs, e.g. peptide, catechol, and all the muscarinic cholinergic receptor subtypes. S/T-->A mutations in analogous i3 domains also prevented receptor internalization in the case of Hm3 and attenuated internalization of Hm2. We propose that an S/T-rich domain in the i3 loop, possibly via phosphorylation reactions, regulates one pathway of GPCR internalization.

Mapping the ligand binding pocket of the human muscarinic cholinergic receptor Hm1: contribution of tyrosine-82.

The ligand binding pocket of many G protein-coupled receptors is thought to be located within the core formed by their seven transmembrane domains (TMDs). Previous results suggested that muscarinic antagonists bind to a pocket located toward the extracellular region of the TMDs, primarily at TMDs 2, 3, 6, and 7. Tyrosine-82 (Y82) is located in TMD2 only one helical turn from the presumed membrane surface of Hm1, whereas a phenylalanine (F124) is found in the equivalent position of the closely related Hm3. In order to determine the contribution of Y82 to Hm1 ligand binding and selectivity versus Hm3, we constructed the point mutation Y82 F of Hm1 and measured binding affinities of various ligands, with 3H-N-methylscopolamine (3H-NMS) as the tracer. The Hm1 wild-type receptor and the Y82F mutant were transfected into human embryonic kidney U293 cells. Whereas the affinities of NMS, carbachol, and atropine were either unchanged (carbachol) or enhanced by less than twofold (atropine and NMS), the affinity of the Hm1-selective pirenzepine was reduced threefold by the Y82F mutation. These changes parallel affinity differences of Hm1 and Hm3, indicating that the Y82 F mutation affects the binding pocket and that Y82 contributes to the binding selectivity among closely related muscarinic receptors.