Transmembrane Domain VI Research Articles

Genomic Characterization of the Lysophosphatidic Acid Receptor Gene, lpA2/Edg4, and Identification of a Frameshift Mutation in a Previously Characterized cDNA

To understand the regulation, evolution, and genetics of lpA2/Edg4, a second lysophosphatidic acid receptor gene, we characterized its complete cDNA sequence, genomic structure, and chromosomal location. The full-length mouse transcript sequence was determined using rapid amplification of cDNA ends. Southern blot and restriction fragment length polymorphism segregation analyses revealed that the mouse gene was present as a single copy and located at the middle of Chromosome 8 near the mutations for myodystrophy (myd) and “kidney–anemia–testes” (kat). This region is syntenic with human chromosome 19p12, where the human genomic clone containing the lpA2 gene (EDG4) was mapped. Sequence analysis of genomic clones demonstrated that both mouse and human transcripts were encoded by three exons, with an intron separating the coding region for transmembrane domain VI. Reverse transcriptase-PCR demonstrated that the three exons were spliced in all mouse tissues shown to express the transcript. Finally, in a comparison of all human lpA2 sequences present in the database, we identified several sequence variants in multiple tumors. One such variant (a G deletion) in the initially characterized Edg4 cDNA clone (derived from an ovarian tumor) results in a frameshift mutation near the 3′ end of the coding region. In addition to increasing our understanding of the mechanisms underlying lysophosphatidic acid signaling and lysophospholipid receptor gene evolution, these results have important implications regarding the genomic targeting and oncogenic potential of lpA2.

Activation of constitutive 5-hydroxytryptamine1B receptor by a series of mutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its Goα protein interactions

Constitutive activity of the recombinant human 5-hydroxytryptamine(1B) (5-HT(1B)) receptor (RC code 2.1.5HT.01.B) was investigated by mutagenesis of the BBXXB motif (in which B represents a basic residue and X a non-basic residue) located in the C-terminal portion of the third intracellular loop. In contrast with wild-type 5-HT(1B) receptors, three receptor mutants (Thr(313)-->Lys, Thr(313)-->Arg and Thr(313)-->Gln) increased their agonist-independent guanosine 5'-[gamma-[(35)S]thio]triphosphate binding response by 26-41%. This activity represented approx. 30% of the maximal response induced by 5-HT and could be reversed by the inverse agonists methiothepin and 3-(3-dimethylaminopropyl)-4-hydroxy-N-(4-pyridin-4-yl phenyl)-benzenamide (GR 55562). Enhanced agonist-independent and agonist-dependent 5-HT(1B) receptor activation was provided by co-expression of a pertussis toxin-resistant rat G(o)alpha Cys(351)-->Ile protein. The wild-type 5-HT(1B) receptor displayed a doubling in basal activity, whereas a spectrum of enhanced basal activities (313-571%) was observed with a series of diverse amino acid substitutions (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, glutamine and arginine) at the 5-HT(1B) receptor position 313 in the presence of pertussis toxin (100 ng/ml). Consequently, the constitutive 5-HT(1B) receptor activity can be modulated by the mutation of Thr(313), and displays a graded range between 11% and 59% of maximal 5-HT(1B) receptor activation by 5-HT. No clear pattern is apparent in the framework of traditionally cited amino acid characteristics (i.e. residue size, charge or hydrophobicity) to explain the observed constitutive activities. The various amino acid substitutions that yielded enhanced activity are unlikely to make similar intramolecular interactions within the 5-HT(1B) receptor. It is hypothesized that the positioning of the junction between the third intracellular loop and transmembrane domain VI is altered by mutation of Thr(313) in the BBXXB motif and thereby unmasks G(alpha)-protein interaction points.

Truncation releases olfactory receptors from the endoplasmic reticulum of heterologous cells.

Olfactory receptors are difficult to express functionally in heterologous cells. We found that olfactory receptors traffic poorly to the plasma membrane even in cells with neuronal phenotypes, including cell lines derived from the olfactory epithelium. Other than mature olfactory receptor neurons, few cells appear able to traffic olfactory receptors to the plasma membrane. In human embryonic kidney 293 cells and Xenopus fibroblasts, olfactory receptor immunoreactivity overlapped with a marker for the endoplasmic reticulum (ER) but not with markers for the Golgi apparatus or endosomes. Except for the ER, olfactory receptors were therefore absent from organelles normally involved in the plasma membrane trafficking of receptors. Olfactory receptors truncated prior to transmembrane domain VI were expressed in the plasma membrane, however. Co-expression of the missing C-terminal fragment with these truncated receptors prevented their expression in the plasma membrane. Intramolecular interactions between N- and C-terminal domains joined by the third cytoplasmic loop appear to be responsible for retention of olfactory receptors in the ER of heterologous cells. Our results are consistent with misfolding of the receptors but could also be explained by altered trafficking of the receptors.

A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function.

Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors.

Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors. The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors. Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor.

Constitutive activation of the human bradykinin B2 receptor induced by mutations in transmembrane helices III and VI.

We report that mutation of specific residues in the human B2 bradykinin (BK) receptor induces its marked constitutive activation, evaluated through inositol phosphate production in COS-7 cells expressing the wild-type or mutant receptors. We provide evidence for a strikingly high constitutive activation of the B2 receptor induced by alanine substitution of the Asn113 residue, located in the third transmembrane domain. These results are reminiscent of our previous finding that mutation of the homologous Asn111 residue induces constitutive activation of the AT1 angiotensin II receptor. BK overstimulation of the constitutively activated mutant N113A receptor was also observed. Phe replacement of the Trp256 residue, fairly conserved in transmembrane domain VI of G protein-coupled receptors, also induced a less prominent but significant constitutive activation. Interestingly, the peptidic HOE 140 compound and an original nonpeptidic compound LF 16 0335, which both behaved as inverse agonists of the wild-type receptor expressed in COS-7 cells, became potent and efficient agonists of the two constitutively activated mutant N113A and W256F receptors. These parallel changes observed for two chemically unrelated series can serve as a basis for future studies of structure-function relationships and modeling of activation processes, based on a detailed analysis of the network of helix-helix interactions, which stabilize the inactive receptor conformation and undergo rearrangements on transition to activated states.

Interspecies Polymorphisms Confer Constitutive Activity to theMastomys Cholecystokinin-B/Gastrin Receptor

The enteroendocrine hormone, gastrin, exerts trophic effects on the gastric mucosa through the CCK-B/gastrin receptor (CCK-BR). To varying degrees in different species, excess circulating gastrin leads to proliferation of enterochromaffin-like cells and to the development of gastric carcinoid tumors. The African rodent, Mastomys natalensis, is distinguished from other mammals by its propensity toward CCK-BR-mediated growth even in the absence of hypergastrinemia. Here, we report that the Mastomys CCK-BR, when expressed in COS-7 cells, differs from the respective human, canine, and rat receptor homologs by its ability to trigger ligand-independent (i.e., constitutive) inositol phosphate formation. To define the molecular basis of this observation, a series of Mastomys-human chimeric receptors was investigated. Functional characterization of these constructs revealed that a limited segment of the Mastomys CCK-BR, transmembrane domain VI through the C-terminal end, is sufficient to confer constitutive activity to the human protein. Mutagenesis studies within this CCK-BR region defined a combination of three Mastomys amino acids that, when introduced into the human receptor, together conferred a level of ligand-independent signaling comparable with the Mastomys CCK-BR. Complementing prior observations that single point mutations can lead to ligand-independent signaling, our findings suggest that multiple naturally occurring amino acid polymorphisms and/or mutations may together result in an enhanced basal level of receptor activity.

Complete cDNA Sequence, Genomic Structure, and Chromosomal Localization of the LPA Receptor Gene,lpA1/vzg-1/Gpcr26

ThelpA1/Gpcr26locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3′ rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5′ sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor,Edg1,but is identical to another more similar orphan receptor (lpA2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 inMus spretus,and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for thevzg-1/lpA1subfamily of receptors compared to those of homologous orphan receptor genes.

Differential Expansion of the N-Formylpeptide Receptor Gene Cluster in Human and Mouse

The human formylpeptide receptor (FPR) gene cluster has three members:FPR1andFPRL1, which are expressed in neutrophils and monocytes and encode seven-transmembrane-domain chemotactic receptors specific for N-formylpeptides, andFPRL2,whose function is unknown. The FPRL1 receptor is also a lipoxin A4 receptor. Using probes for the three human genes we have cloned six distinct mouse genes, designatedFpr1andFpr-rs1throughFpr-rs5,which form a cluster on chromosome 17 in a region of conserved synteny with human chromosome 19.Fpr1encodes a functional receptor and is clearly the orthologue ofFPR1.BothFpr-rs1andFpr-rs2have higher sequence homology toFPRL1than toFPRL2; Fpr-rs1is 97% identical in amino acid sequence to a previously reported cDNA that encodes a lipoxin A4 receptor, whereas the putative ligand forFpr-rs2is unknown.Fpr-rs3, Fpr-rs4,andFpr-rs5appear to lack human counterparts and are most similar in sequence toFPRL1.RNA forFpr1, Fpr-rs1,andFpr-rs2is present in leukocytes, spleen, and lung, whereas RNA forFpr-rs3was detected only in skeletal muscle. We did not detectFpr-rs4orFpr-rs5RNA in any tissue tested. Moreover,Fpr-rs5has a stop codon in the protein-coding region corresponding to transmembrane domain VI and may not encode a functional receptor. These results suggest that the FPR gene cluster has undergone differential expansion in mammals withFPRL2, Fpr-rs2, Fpr-rs3, Fpr-rs4,andFpr-rs5arising after divergence of human and mouse.

Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.

Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

Mutations of thyrotropin receptor gene

Thyrotropin is the primary pituitary hormone which stimulates the growth and differentiation of thyroid cells. TSH binds a specific receptor present in the plasma membrane of thyroid cells and signals the G protein transducers, which activate different effectors, mainly adenyl cyclase and phospholipase C. The TSH receptor belongs to a broad class of receptors known as seven-loop receptors because they contain a long stretch of amino acids which cross the plasma membrane seven times. Mutations in the TSH receptor gene have been found in hyperfunctioning thyroid adenomas. These mutations are: (a) somatic (present only in the tumor), (b) dominant (only one copy of the gene is affected), and (c) lead to the constitutive activation of the cAMP signaling cascade. Most mutations which have been identified occur in the intracellular loop III and in the transmembrane domain VI. Germline mutations in the same regions of the receptor have been found in congenital nonautoimmune hyperthyroidism. In addition, germ line mutations have been described in the extracellular domain of the receptor leading to increased TSH levels. The clinical implications of these findings are discussed.

Identification in the rat neurotensin receptor of amino-acid residues critical for the binding of neurotensin.

In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the receptor either did not affect (125)I-Tyr3-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids Asp113 in the second transmembrane domain (TM) and of Arg149 or Asp150 in TM III yielded receptors that bound NT as efficiently as the native receptor. By contrast, replacement of the Asp139 residue in the 1st extracellular loop, or of Arg143 or Arg327-Arg328 residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp139-Gly- and Arg143-Gly-modified receptors remained functional in spite of a lowered response to NT whereas the Arg327-Arg328 mutant form was totally insensitive to NT at concentrations up to 10 microM. In the case of the Arg327-Arg328 mutation, the observed insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp139 and Arg143 residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin.

A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation

One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F.(1990) Nature 348, 125-132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that beta2-adrenergic receptors do form SDS-resistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits beta-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.

Arrangement of Transmembrane Domains in Adrenergic Receptors: SIMILARITY TO BACTERIORHODOPSIN

G protein-coupled receptors (GPCRs) have seven hydrophobic domains, which are thought to span the lipid bilayer as alpha helical transmembrane domains (TMDs). The tertiary structure of GPCRs has not been determined; however, molecular models of GPCRs have generally been based on bacteriorhodopsin, which is functionally unrelated to GPCRs but has a similar secondary structure. We sought to examine the validity of using bacteriorhodopsin as a scaffold for GPCR model building by experimentally determining the orientation of the TMDs of adrenergic receptors in the plasma membrane. In separate experiments, three sequential amino acid residues (Leu-310, Leu-311, Asn-312) in TMD VII of the beta 2 adrenoreceptors were mutated to the amino acids found in the homologous domain of the alpha 2 adrenoceptor (Phe, Phe, Phe). Exchange of Asn-312 and Leu-311 in the beta 2 adrenoceptor resulted in nonfunctional proteins, most likely due to incompatibility of the introduced bulky phenylalanine side chain with adjacent structural domains in the beta 2 adrenoreceptor. This structural incompatibility was "repaired" by replacing the specific beta 2 TMD sequence with an alpha 2 receptor sequence. TMD I and TMD II complemented the Asn-312-->Phe mutation, and TMD III and TMD VI complemented the Leu-311-->Phe mutation. These results indicate that TMDs I, II, III, and VI surround TMD VII in a counter-clockwise orientation analogous to the orientation of TMDs in bacteriorhodopsin.

Delineation of the peptide binding site of the human galanin receptor.

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th

Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling.

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.

Identification and functional characterization of two new somatic mutations causing constitutive activation of the thyrotropin receptor in hyperfunctioning autonomous adenomas of the thyroid.

It has recently been shown that somatic and germ line mutations of the TSH receptor gene cause autonomous hyperfunctioning thyroid adenomas and nonautoimmune toxic thyroid hyperplasia by constitutive activation of the TSH receptor. A "saturated" map of these mutations is a prerequisite for a systematic screening for these clinically important mutations. In this context, it is also of interest to determine whether different amino acid substitutions at the same residue cause constitutive activation of the TSH receptor, as suggested by site-directed mutagenesis of the alpha 1 beta-adrenergic receptor. We, therefore, screened further hyperfunctioning autonomous adenomas of the thyroid for constitutively activating mutations. We identified two new somatic mutations, changing alanine in position 623 to valine (A623V) and threonine in position 632 to isoleucine (T632I). Both mutations constitutively activated cAMP when transiently expressed in COS cells. Together with neighboring mutations, the T632I mutation demonstrates the importance of transmembrane domain VI for the activation of the TSH receptor and characterizes it as a hot spot for constitutively activating mutations. The previously identified A623I and the newly identified A623V mutations demonstrate that several amino acid substitutions at the same residue can cause constitutive activation of the TSH receptor.

Chimeric D1/D2 dopamine receptors. Distinct determinants of selective efficacy, potency, and signal transduction.

D1/D2 chimeras were constructed that had D1 dopamine receptor sequence at the amino-terminal end and D2 dopamine receptor sequence at the carboxyl-terminal end. The chimeras with the first four, five and six transmembrane domains of the D1 receptor (CH2, CH3, CH4, respectively) bound the D1 receptor antagonist [3H]SCH 23390 with high affinity. Reciprocal chimeras constructed with D2 receptor sequence at the amino-terminal end displayed no detectable specific binding of [3H]SCH 23390, [125I]epidepride, or [3H]spiperone. CH2, CH3, and CH4 had lower affinity than either D1 or D2 dopamine receptors for the nonselective antagonists and agonists and D2-selective antagonists tested. The chimeric receptors had affinities for three D1-selective ligands and the D2-selective agonist, quinpirole, that were intermediate between D1 and D2 receptor affinities for the drugs. The substantial loss or gain of affinity for three ligands upon replacement of D1 transmembrane VII with D2 sequence (CH4) suggests an important role for this region in the selectivity of these drugs. Stimulation of adenylyl cyclase activity by D1 agonists occurred in cells expressing CH3 and CH4, both of which included the D1 third cytoplasmic loop, but not in cells expressing CH1 or CH2, both with the D2 third cytoplasmic loop. However, only CH3 was able to mediate stimulation of adenylyl cyclase by quinpirole, implying that D2 receptor transmembrane domain VI was an important determinant of the selective efficacy of quinpirole. On the other hand, transmembrane domain VII was particularly important for the selective potency of quinpirole. Inhibition of beta-adrenergic receptor-stimulated adenylyl cyclase activity by dopamine was seen in cells expressing D2 receptors and CH1, but not CH2, CH3, or CH4. Thus, the third cytoplasmic loop of D1 dopamine receptors was crucial for the coupling of the receptors to Gs, but inhibition of adenylyl cyclase via Gi required structural features, such as the second cytoplasmic loop of the D2 receptor, in addition to the 3rd cytoplasmic loop.

-mu opiate receptor. Charged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity.

The mu opiate receptor is a principal brain site for activities of morphine, other opiate drugs, and opioid peptides in modulating pain and altering mood. Recent cloning of cDNAs encoding rat and human mu receptors reveals charged amino acid residues within putative transmembrane domains (TMs) II, III, and VI, a substantial N-terminal extracellular domain, and a C-terminal intracellular domain. Deletion of 64 N-terminal amino acids produced little effect on receptor function (Wang, J.B., Imai, Y., Eppler, C.M., Gregor, P., Spivak, C.E., and Uhl, G.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10230-10234). Further deletion of 33 C-terminal amino acids yielded a receptor at which morphine, but not the substituted enkephalin DAMGO ([D-Ala2,MePhe4,Glyol5]enkephalin), inhibited adenylate cyclase. Alanine substitution for each charged TM residue in the N-terminally deleted receptor reduced affinities for morphine, DAMGO, and the opiate antagonist naloxone. Replacement of TM II Asp114 with asparagine or glutamic acid increased mu receptor affinity for naloxone. TM II and TM III glutamic acid substitutions for Asp114 and Asp147 reduced agonist binding affinities but allowed full inhibition of adenylate cyclase at high agonist concentrations. TM VI histidine substitution with alanine yielded a receptor that produced almost twice the cyclase inhibition displayed by the wild type receptor in parallel transient expression assays. These findings underscore the importance of charged residues in TM II, III, and VI for different receptor functions and the modest involvement of extensive portions of N- and C-terminal receptor domains in these processes.

Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors.

The molecular mechanisms through which muscarinic receptors are activated upon binding of the neurotransmitter acetylcholine (ACh) are still poorly understood. Classical structure-function relationship studies have previously established that the ACh ester moiety plays a key role in muscarinic receptor recognition and activation. Consistent with this notion, all recently proposed three-dimensional muscarinic receptor models predict that an asparagine residue present in transmembrane domain VI of all muscarinic receptors is critically involved in the binding of the ACh ester moiety by means of hydrogen bonding. To test the correctness of this hypothesis, we created several mutant m3 muscarinic receptors in which this residue (Asn507) was replaced with alanine, serine, or aspartic acid. Radioligand binding studies with transfected COS-7 cells showed that, in contrast to the predictions made based on molecular modeling studies, all three mutant receptors were able to bind ACh and the structurally related muscarinic agonist, carbachol, with high affinities which differed from the corresponding wild type values by less than 5-fold. However, all three mutations led to dramatic reductions (235-28,300-fold) in binding affinities for certain subclasses of muscarinic antagonists including atropine-like agents and pirenzepine. The m3(Asn507-->Ala) and m3(Asn507-->Asp) mutant receptors were able to mediate carbachol-induced phosphatidylinositol hydrolysis in a fashion similar to that of the wild type receptor. Interestingly, the m3(Asn507-->Ser) mutant receptor displayed about 2-fold increased basal inositol phosphate levels, raising the possibility that it is constitutively active. In conclusion, our data suggest that the asparagine residue present in transmembrane domain VI of all muscarinic receptors is not critical for ACh binding and agonist-induced receptor activation, but plays a key role in the binding of certain subclasses of muscarinic antagonists.