Biomedical subjects

Graeme Milligan

Publications and source records attributed to Graeme Milligan.

At least 19 recordsLinked to original sources

PubMed2006

Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function.

Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonist-independent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers.

Base Sequence↗

PubMed2006

Oligomeric structure of the alpha1b-adrenoceptor: comparisons with rhodopsin.

The structural basis of the quaternary organization of rhodopsin has recently been explored and modeled. Because information obtained from studying rhodopsin has frequently been directly applicable to other G protein-coupled receptors we wished to ascertain if dimeric and/or oligomeric forms of the alpha(1b)-adrenoceptor could be observed and if so whether rhodopsin might provide insights into the quaternary structure of this receptor. Co-immunoprecipitation and both conventional and time-resolved fluorescence resonance energy transfer studies demonstrated quaternary structure of the alpha(1b)-adrenoceptor and, in concert with the reconstitution of fragments of this receptor, provided information on the molecular basis of these interactions. Development of three color fluorescence resonance energy transfer (FRET) allowed the imaging of alpha(1b)-adrenoceptor oligomers in single living cells. Mutation of hydrophobic residues in transmembrane domains I and IV of the receptor resulted in marked reduction in three color FRET suggesting an alteration in oligomeric organization and potential similarities with rhodopsin. The mutated alpha(1b)-adrenoceptor was unable to reach the cell surface, did not become terminally N-glycosylated and was unable to signal.

Animals↗

PubMed2006

Interaction of neurochondrin with the melanin-concentrating hormone receptor 1 interferes with G protein-coupled signal transduction but not agonist-mediated internalization.

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Galpha(i/o) and Galpha(q/11) family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K(+) channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [(35)S]guanosine 5'-3-O-(thio)triphosphate to both Galpha(o1) and Galpha(11). Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.

Amino Acid Sequence↗

PubMed2006

Mu-delta opioid receptor functional interaction: Insight using receptor-G protein fusions.

Fusion proteins between a receptor and a pertussis toxin-insensitive G(i)alpha subunit were used to gain insight into the molecular interactions that take place upon mu and delta opioid receptor heterodimerization. When mu opioid receptor-G(i1)alpha fusions were coexpressed with nonfused delta opioid receptors in human embryonic kidney 293 cells, or vice versa, receptor heterodimers were detected by coimmunoprecipitation. In pertussis toxin-treated cells, receptor coexpression decreased the amount of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) incorporated in the fused G alpha protein after the addition of agonists specific for the receptor-G(i1)alpha fusion. In addition, activation of the G alpha protein occurred in heterodimers upon addition of an agonist specific for the nonfused receptor. It remained unaffected by an inverse agonist specific for the receptor-G(i1)alpha fusion. These data suggest that signaling through the receptor-G(i1)alpha fusion protein is impaired in heterodimers and support a mechanism in which activation of the G alpha subunit is promoted by a direct interaction with the nonfused receptor. Alternatively, receptor coexpression did not modify the ligand binding properties for the high-affinity state of the receptor-G(i1)alpha fusion nor the EC50 values for agonist-induced [35S]GTPgammaS incorporation in the G(i1)alpha subunit. In addition, no binding competition was observed between delta and mu ligands. Together, the data point to mu-delta opioid receptor heterodimers formed by contact interactions between monomers that retain their structural integrity.

Analgesics, Opioid↗

PubMed2006

Up-regulation of the angiotensin II type 1 receptor by the MAS proto-oncogene is due to constitutive activation of Gq/G11 by MAS.

Coexpression of the MAS proto-oncogene with the angiotensin II type 1 (AT(1)) receptor in CHO-K1 cells has been reported to increase the number of [(3)H]angiotensin II-binding sites, although MAS does not bind [(3)H]angiotensin II. In HEK293 cells stably expressing AT(1) receptor-cyan fluorescent protein (CFP), MAS-yellow fluorescent protein (YFP) expression from an inducible locus caused strong up-regulation of AT(1) receptor-CFP amounts and [(3)H]angiotensin II binding levels. The time course of AT(1) receptor-CFP up-regulation was also markedly slower than that of induction of MAS expression. These effects were not mimicked by induced expression of I138D MAS-YFP, a mutant unable to cause constitutive loading of [(35)S]guanosine 5'-O-(thiotriphosphate) onto the phospholipase Cbeta-linked G protein Galpha(11). Protein kinase C (PKC) inhibitors and the selective Galpha(q)/Galpha(11) inhibitor YM-254890 fully blocked MAS-induced up-regulation of AT(1) receptor-CFP amounts, whereas the PKC activator phorbol 12-myristate 13-acetate produced strong up-regulation of AT(1) receptor-CFP without induction of MAS-YFP expression and in the presence of I138D MAS-YFP. The C-terminal tail of the AT(1) receptor is a known target for PKC-mediated phosphorylation. In cells stably expressing a C-terminally truncated version of the AT receptor, induction of MAS expression did not up-regulate the truncated construct levels. These data demonstrate that the ability of MAS to up-regulate AT(1) receptor levels reflects the constitutive capacity of MAS to activate Galpha(q)/Galpha(11) and hence stimulate PKC-dependent phosphorylation of the AT(1) receptor.

Animals↗

PubMed2006

G protein-coupled receptors for free fatty acids.

Free fatty acids (FFAs) are not only an important direct source of energy but they also play key roles in regulating a range of physiological responses. Although it was long assumed that such effects of FFAs must occur following cellular uptake, and potentially via their conversion to fatty acyl-CoAs, it is now apparent that FFAs also function directly as agonists at a number of G protein-coupled receptors (GPCRs). Tissue distribution studies and, subsequently, siRNA knock-down experiments have indicated key roles for these GPCRs in glucose homeostasis, adipogenesis, white cell recruitment and potentially in a range of other processes. Considerable interest is thus now centred on the generation of potent and selective small molecule ligands, both as tool compounds to further unravel the biology and physiological role of this group of GPCRs and as starting points for possible therapeutic intervention in a range of areas, particularly those associated with 'metabolic syndrome'.

Animals↗

PubMed2006

Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1-A2A receptor heteromers.

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Animals↗

PubMed2006

The chemokine receptor CCX-CKR mediates effective scavenging of CCL19 in vitro.

The chemokines CCL19, CCL21 and CCL25, by signalling through the receptors CCR7 or CCR9, play critical roles in leukocyte homing. They also bind another heptahelical surface protein, CCX-CKR. CCX-CKR cannot couple to typical chemokine receptor signalling pathways or mediate chemotaxis, and its function remains unclear. We have proposed that it controls chemokine bioavailability. Here, using transfected HEK293 cells, we have shown that both CCX-CKR and CCR7 mediate rapid CCL19 internalisation upon initial chemokine exposure. However, internalised CCL19 was more efficiently retained and degraded after uptake via CCX-CKR. More importantly, CCR7 rapidly became refractory for CCL19 uptake, but the sequestration activity of CCX-CKR was enhanced. These properties endowed CCX-CKR with an impressive ability to mediate progressive sequestration and degradation of large quantities of CCL19, and conversely, prevented CCR7-expressing cells from extensively altering their chemokine environment. These differences may be linked to the routes of endocytosis used by these receptors. CCX-CKR, unlike CCR7, was not critically dependent on beta-arrestins or clathrin-coated pits. However, over-expression of caveolin-1, which stabilises caveolae, blocked CCL19 uptake by CCX-CKR while having no impact on other chemokine receptors, including CCR7. These data predict that CCX-CKR scavenges extracellular chemokines in vivo to modify responses through CCR7.

Cell Line↗

PubMed2006

G-protein-coupled receptor heterodimers: pharmacology, function and relevance to drug discovery.

The growing recognition that members of the rhodopsin-like family A G-protein-coupled receptors (GPCRs) exist and function as dimers or higher-order oligomers, and that GPCR hetero-dimers and -oligomers are present in physiological tissues, offers novel opportunities for drug discovery. Differential pharmacology, function and regulation of GPCR hetero-dimers and -oligomers suggest means to selectively target GPCRs in different tissues and hint that the mechanism of function of several pharmacological agents might be different in vivo than anticipated from simple ligand-screening programmes that rely on heterologous expression of a single GPCR.

Animals↗

PubMed2006

Heterotrimeric G-proteins: a short history.

Some 865 genes in man encode G-protein-coupled receptors (GPCRs). The heterotrimeric guanine nucleotide-binding proteins (G-proteins) function to transduce signals from this vast panoply of receptors to effector systems including ion channels and enzymes that alter the rate of production, release or degradation of intracellular second messengers. However, it was not until the 1970s that the existence of such transducing proteins was even seriously suggested. Combinations of bacterial toxins that mediate their effects via covalent modification of the alpha-subunit of certain G-proteins and mutant cell lines that fail to generate cyclic AMP in response to agonists because they either fail to express or express a malfunctional G-protein allowed their identification and purification. Subsequent to initial cloning efforts, cloning by homology has defined the human G-proteins to derive from 35 genes, 16 encoding alpha-subunits, five beta and 14 gamma. All function as guanine nucleotide exchange on-off switches and are mechanistically similar to other proteins that are enzymic GTPases. Although not readily accepted initially, it is now well established that beta/gamma complexes mediate as least as many functions as the alpha-subunits. The generation of chimeras between different alpha-subunits defined the role of different sections of the primary/secondary sequence and crystal structures and cocrystals with interacting proteins have given detailed understanding of their molecular structure and basis of function. Finally, further modifications of such chimeras have generated a range of G-protein alpha-subunits with greater promiscuity to interact across GPCR classes and initiated the use of such modified G-proteins in drug discovery programmes.

Amino Acid Sequence↗

PubMed2006

Carvedilol-induced antagonism of angiotensin II: a matter of alpha1-adrenoceptor blockade.

OBJECTIVE: To investigate whether renin-angiotensin system blockade might underlie the favorable metabolic effects of the nonselective beta + alpha1-adrenoceptor blocker carvedilol as compared with the selective beta1-adrenoceptor blocker metoprolol. METHODS: Human coronary microarteries (HCMAs), obtained from 32 heart valve donors, were mounted in myographs. RESULTS: Angiotensin II and the alpha1-adrenoceptor agonist phenylephrine constricted HCMAs to maximally 63 +/- 10 and 46 +/- 15% of the contraction to 100 mmol/l K. Neither carvedilol, metoprolol, the nonselective beta-adrenoceptor antagonist propranolol, nor the alpha1-adrenoceptor antagonist prazosin affected the constrictor response to angiotensin II. alpha1-adrenoreceptors and beta-adrenoceptors are thus not involved in the direct constrictor effects of angiotensin II. When added to the organ bath at a subthreshold concentration, angiotensin II greatly amplified the response to phenylephrine. Both carvedilol and the angiotensin II type 1 (AT1) receptor antagonist irbesartan inhibited this angiotensin II-induced potentiation. Furthermore, carvedilol blocked the angiotensin II-induced amplification of phenylephrine-induced inositol phosphate accumulation in cardiomyocytes. CONCLUSIONS: AT1-alpha1-receptor crosstalk, involving inositol phosphates, sensitizes HCMAs to alpha1-adrenoceptor agonists. Our results suggest that, in the presence of an increased sympathetic tone, carvedilol provides AT1 receptor blockade via its alpha1-adrenoceptor blocking effects. This could explain the favorable effects of carvedilol versus metoprolol.

Adolescent↗

PubMed2005

Functional interactions between the alpha1b-adrenoceptor and Galpha11 are compromised by de-palmitoylation of the G protein but not of the receptor.

Both the alpha1b-adrenoceptor and Galpha11 are targets for post-translational thio-acylation that is regulated by agonist occupancy of the receptor [P.A. Stevens, J. Pediani, J.J. Carrillo, G. Milligan, J. Biol. Chem. 276 (2001) 35883]. In co-expression studies mutation of the sites of thio-acylation in the G protein or treatment of cell membranes with hydroxylamine greatly reduced agonist stimulation of guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTPgammaS) binding. In alpha1b-adrenoceptor-Galpha11 fusion proteins mutation of thio-acylation sites in receptor or G protein did not alter the binding affinity of the antagonist [3H]prazosin or the agonist phenylephrine. Although the potency of phenylephrine to stimulate binding of [35S]GTPgammaS to alpha1b-adrenoceptor-Galpha11 fusion proteins was unaffected by the thio-acylation potential of either element, the maximal effect was reduced by some 50% when the G protein but not the receptor was mutated to prevent thio-acylation. This reflected lack of thio-acylation of the G protein rather than mutation of Cys9 and Cys10 to Ser because treatment with hydroxylamine mimicked this in fusions containing the wild type G protein but was without effect in those mutated to prevent thio-acylation. Mutation to reduce binding of beta/gamma to Galpha11 markedly reduced phenylephrine stimulation of [35S]GTPgammaS binding. Combination of mutations to prevent thio-acylation and beta/gamma binding did not, however, have an additive effect on [35S]GTPgammaS binding. These results indicate that the thio-acylation status of the alpha1b-adrenoceptor does not regulate G protein activation whereas thio-acylation of Galpha11 plays a key role in activation by the receptor beyond providing membrane association and proximity.

Acylation↗

PubMed2005

Interactions between the Mas-related receptors MrgD and MrgE alter signalling and trafficking of MrgD.

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to beta-alanine but not L-alanine by elevating intracellular [Ca(2+)], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to beta-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of beta-alanine to phosphorylate ERK1 and ERK2 as well as maintaining the capacity of beta-alanine to elevate intracellular [Ca(2+)], which was reduced in magnitude and slowed in response with increasing times of expression of rMrgD in isolation. Associated with these effects, the presence of rMrgE restricted beta-alanine-induced internalization of rMrgD. This is the first report of heterodimeric interactions between members of the Mas-related gene (Mrg) receptor family and indicates that interactions between rMrgD and rMrgE modulate the function of rMrgD. Because the Mrg receptors are potential therapeutic targets in pain, these results suggest that efforts to understand the function and regulation of individual Mrg family receptors may require coexpression of relevant pairs.

Animals↗

PubMed2005

Techniques: promiscuous Galpha proteins in basic research and drug discovery.

Assay technologies that measure the activation of heterotrimeric (alphabetagamma) G proteins by G-protein-coupled receptors (GPCRs) are well established within the pharmaceutical industry, either for pharmacological characterization or for the identification of natural or surrogate receptor ligands. Despite recent evidence indicating that GPCR-linked signalling events might not be mediated exclusively by G proteins, G-protein activation remains a common benchmark for assessing GPCR family members. Thus, assay systems that translate ligand-mediated modulation of GPCRs into G-protein-dependent intracellular responses still represent key components of both basic research and the drug discovery process. In this article, the current knowledge and recent progress of integrating Galpha subunits into assay systems for GPCR drug discovery will be reviewed. Emphasis is given to novel promiscuous and chimeric Galpha proteins. Because of their ability to interact with a wide range of GPCRs, such novel G proteins are likely to be incorporated rapidly into drug discovery programmes.

Amino Acid Sequence↗

PubMed2005

A key serine for the GTPase-activating protein function of regulator of G protein signaling proteins is not a general target for 14-3-3 interactions.

Mammalian regulator of G protein signaling (RGS) proteins are highly conserved within the RGS domain. Of amino acids that are universal, a serine residue at the C terminus of this domain has been described as the binding site in RGS7 for 14-3-3 proteins. However, studies with the related RGS3 indicate that the site of interaction is not within the RGS domain. We confirm that the interaction of RGS3 with 14-3-3tau and 14-3-3zeta requires Ser264 and not the RGS domain and show both that mutation of the conserved RGS domain serine, Ser496 in RGS3, to either alanine or aspartate does not prevent binding of 14-3-3 proteins and that 14-3-3 proteins do not inhibit GTPase-activating protein (GAP) activity against receptor-activated Galpha(o1). However, mutation of Ser496 does directly impair the action of RGS3 as a GAP against receptor-activated Galpha(o1). We mutated the equivalent serine residue in the family B/R4 RGS proteins RGS1 and RGS16. Using two distinct assay formats, conversion to aspartate virtually abolished GAP activity, whereas conversion to alanine decreased potency 20-fold. Neither alteration modulated interactions with 14-3-3tau or 14-3-3zeta, but the 14-3-3 proteins did not modulate the GAP activity of the wild-type or mutant RGS proteins. Although interactions between 14-3-3 proteins and many RGS proteins can be observed, this does not involve this conserved serine and does not inherently modify GAP function.

14-3-3 Proteins↗

PubMed2005

Functional complementation and the analysis of opioid receptor homodimerization.

Complementation of function after coexpression of pairs of nonfunctional G protein-coupled receptors that contain distinct inactivating mutations supports the hypothesis that such receptors exist as dimers. Chimeras between members of the metabotropic glutamate receptor-like family have been particularly useful because the N-terminal ligand binding and heptahelical transmembrane elements can be considered distinct domains. To examine the utility of a related approach for opioid receptors, fusion proteins were generated in which a pertussis toxin-resistant (Cys351Ile) variant of the G protein Gi1alpha was linked to the C-terminal tails of the delta opioid peptide (DOP), kappa opioid peptide, and mu opioid peptide receptors. Each was functional as measured by agonist stimulation of guanosine 5'-([gamma-35S]thio)triphosphate ([35S]GTPgammaS) binding in Gialpha immunoprecipitates from membranes of pertussis toxin-treated HEK293 cells. Agonist function was eliminated either by fusion of the receptors to Gi1alphaGly202Ala,Cys351Ile or mutation of a pair of conserved Val residues in intracellular loop 2 of each receptor. Coexpression, but not simple mixing, of the two inactive fusion proteins reconstituted agonist-loading of [35S]GTPgammaS for each receptor. At equimolar amounts, reconstitution of DOP receptor function was more extensive than kappa or mu opioid receptor. Reconstitution of DOP function required two intact receptors and was not achieved by provision of extra Gi1alphaCys351Ile membrane anchored by linkage to DOP transmembrane domain 1. Inactive forms of all G protein alpha subunits can be produced by mutations equivalent to Gi1alphaGly202Ala. Because the amino acids modified in the opioid receptors are highly conserved in most rhodopsin-like receptors, this approach should be widely applicable to study the existence and molecular basis of receptor dimerization.

Base Sequence↗

PubMed2005

The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities.

Both homo- and heterodimeric interactions between the CXCR1 and CXCR2 chemokine receptors were observed following co-expression of forms of these receptors in HEK293 cells using assays, including co-immunoprecipitation, single cell imaging of fluorescence resonance energy transfer, cell surface time-resolved fluorescence resonance energy transfer, and bioluminescence resonance energy transfer. These interactions were constitutive and unaffected by the presence of the agonist interleukin 8 and selective as no significant interactions were noted between either the CXCR1 or CXCR2 receptor and the alpha(1A)-adrenoreceptor. Saturation bioluminescence resonance energy transfer indicated that heteromeric interactions between CXCR1 and CXCR2 were of similar affinity as the corresponding homomeric interactions. A novel endoplasmic reticulum trapping strategy demonstrated that these interactions were initiated during protein synthesis and maturation and prior to cell surface delivery. These studies indicated that CXCR1-CXCR2 heterodimers are as likely to form in cells co-expressing these two chemokine receptors as the corresponding homodimers and stand in contrast to previous studies indicating an inability of the CXCR1 receptor to homodimerize or to interact with the CXCR2 receptor (Trettel, F., Di Bartolomeo, S., Lauro, C., Catalano, M., Ciotti, M. T., and Limatola, C. (2003) J. Biol. Chem. 278, 40980-40988).

Base Sequence↗

PubMed2005

The sustainability of interactions between the orexin-1 receptor and beta-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation.

The orexin-1 receptor interacts with beta-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with beta-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of beta-arrestin-2-GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with beta-arrestin-2, studies in wild-type and beta-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a beta-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with beta-arrestin-2, and indicate an important role of beta-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

Amino Acid Sequence↗