Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer.

Dimerization of some G protein-coupled receptors has recently been demonstrated, but how widespread this phenomenon might be and its functional implications are not yet clear. We have utilized biophysical and biochemical techniques to evaluate whether the type A cholecystokinin (CCK) receptor can form oligomeric complexes in the plasma membrane and the impact of ligand binding and signaling on such complexes. We investigated the possibility of bioluminescence resonance energy transfer (BRET) between receptor constructs that included carboxyl-terminal tags of Renilla luciferase or yellow fluorescent protein. Indeed, co-expression of these constructs in COS cells resulted in the constitutive presence of a significant BRET signal above that in a series of controls, with this signal reduced by co-expression of competing non-tagged CCK receptors. The presence of an oligomeric complex of CCK receptor molecules was confirmed in co-immunoprecipitation experiments. Occupation of CCK receptors with agonist ligands (CCK or gastrin-4) resulted in the rapid reduction in BRET signal in contrast to the enhancement of such a signal reported after agonist occupation of the beta(2)-adrenergic receptor. These effects on CCK receptor oligomerization were concentration-dependent, correlating with the potencies of the agonists. A smaller effect was observed for a partial agonist, and no effect was observed for antagonist occupation of this receptor. Agonist-induced reduction in BRET signal was also observed for pairs of CCK receptors with a donor-acceptor pair situated in other positions within the receptor. Manipulation of the phosphorylation state of CCK receptor using protein kinase C activation with phorbol ester or inhibition with staurosporine had no effect on the basal level or agonist effect on CCK receptor oligomerization. This provides the first evidence for CCK receptor oligomerization in living cells, with insights that the active conformation of this receptor dissociates these complexes in a phosphorylation-independent manner.     

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

Agonist-stimulatedphosphorylation of guanine nucleotide-binding protein (Gprotein)-coupled receptors has been recognized as an importantmechanism for desensitization by interfering with coupling of theactivated receptor with its G protein. We recently described a mutantof the CCK receptor that modified two of five key sites ofphosphorylation (S260,264A) and eliminated agonist-stimulated receptorphosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant hadimpaired rapid desensitization but was ultimately able to bedesensitized by normal receptor internalization. Here we demonstratethat this mutant receptor is also defective in resensitization, withabnormal recycling to the cell surface. To explore this, anotherreceptor mutant was prepared, replacing the same serines withaspartates to mimic the charge of serine-phosphate (S260,264D). Thismutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. Itwas internalized like wild-type receptors and was resensitized andtrafficked normally. This provides evidence for an additional importantfunction for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor toexpose other potential sites of phosphorylation and to expose domainsinvolved in the targeting and trafficking of endosomes. Thehierarchical phosphorylation of these sites may play a key role inreceptor regulation.

     

Dual pathways of internalization of the cholecystokinin receptor

Receptor molecules play a major role in the desensitization of agonist- stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.     

Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization

Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schr?der, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., H?llt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.     

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.     

Distinct molecular mechanisms for agonist peptide binding to types A and B cholecystokinin receptors demonstrated using fluorescence spectroscopy

Fluorescence spectroscopy provides a direct method for evaluating the environment of a fluorescent ligand bound to its receptor. We utilized this methodology to determine the environment of Alexa within a cholecystokinin (CCK)-like probe (Alexa488-Gly-[(Nle(28,31))CCK-26-33]; CCK-8 probe) bound to the type A CCK receptor (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Here, we study this probe at the type B CCK receptor and develop another probe with its fluorophore closer to the carboxyl-terminal pharmacophore of type B receptor ligands (Alexa488-Trp-Nle-Asp-Phe-NH2; CCK-4 probe). Both probes bound to type B CCK receptors in a saturable and specific manner and represented full agonists. Similar to the type A receptor, at the type B receptor these probes exhibited shorter lifetimes and lower anisotropy when the receptor was in the active conformation than when it was shifted to its inactive, G protein-uncoupled state using guanosine 5'-[beta,gamma-imido]-triphosphate trisodium salt. Absolute values for lifetime and anisotropy were lower for the CCK-8 probe bound to the type B receptor than for this probe bound to the type A receptor, and Alexa fluorescence was more easily quenched by iodide at the type B receptor. This represents the first direct evidence that, despite having identical affinities for binding and potencies for activating type A and B receptors, CCK is docked via distinct mechanisms, with the amino terminus more exposed to the aqueous milieu when bound to the type B CCK receptor than to the type A CCK receptor. Of interest, despite this difference in binding, activation of both receptors results in analogous direction of movement of the fluorescent indicator probes.     

Olfactory receptor localization and function: an emerging role for GPCR heterodimerization

Research on olfaction has been fraught with considerable frustration because none of the hundreds of olfactory receptors make it to the cell surface on their own when expressed in heterologous systems. Recent work indicates that the heterodimerization of olfactory receptors with beta2-adrenergic receptors results in surface expression of these G protein-coupled receptors. Similar conclusions--that heterodimerization is essential for surface expression of olfactory receptors--have been drawn from research in Drosophila utilizing completely different knockout and functional approaches. Together these findings may unlock the solution to a problem that has plagued the molecular study of olfaction since the cloning of the first olfactory G protein-coupled receptor over twelve years ago.     

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1) and small heterodimer partner (SHP) (NR0B2) form homodimers individually, as well as DAX1-SHP heterodimers

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1), and small heterodimer partner (SHP) (NR0B2) are atypical nuclear receptor superfamily members that function primarily as corepressors through heterodimeric interactions with other nuclear receptors. Mutations in DAX1 cause adrenal hypoplasia congenita, and mutations in SHP lead to mild obesity and insulin resistance, but the mechanisms are unclear. We investigated the existence and subcellular localization of DAX1 and SHP homodimers and the dynamics of homodimerization. We demonstrated DAX1 homodimerization in the nucleus and cytoplasm, and dissociation of DAX1 homodimers upon heterodimerization with steroidogenic factor 1 (SF1) or ligand-activated estrogen receptor-alpha (ERalpha). DAX1 homodimerization involved an interaction between its amino and carboxy termini involving its LXXLL motifs and activation function (AF)-2 domain. We observed SHP homodimerization in the nucleus of mammalian cells and showed dissociation of SHP homodimers upon heterodimerization with ligand-activated ERalpha. We observed DAX1-SHP heterodimerization in the nucleus of mammalian cells and demonstrated the involvement of the LXXLL motifs and AF-2 domain of DAX1 in this interaction. We further demonstrate heterodimerization of DAX1 with its alternatively spliced isoform, DAX1A. This is the first evidence of homodimerization of individual members of the unusual NR0B nuclear receptor family and heterodimerization between its members. Our results suggest that DAX1 forms antiparallel homodimers through the LXXLL motifs and AF-2 domain. These homodimers may function as holding reservoirs in the absence of heterodimeric partners. The formation of DAX1 and SHP homodimers and DAX1-SHP and DAX1-DAX1A heterodimers suggests the possibility of novel functions independent of their coregulator roles, suggesting additional complexity in the molecular mechanisms of DAX1 and SHP action.     

G protein-coupled receptor kinase-mediated desensitization of metabotropic glutamate receptor 1A protects against cell death

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.     

Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A)

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst(2A) and sst(3) exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and sst(3). The sst(2A)-sst(3) heterodimer exhibited high affinity binding to somatostatin-14 and the sst(2)-selective ligand L-779,976 but not to the sst(3)-selective ligand L-796,778. Like the sst(2A) homodimer, the sst(2A)-sst(3) heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst(2)-selective ligand L-779,976. However, unlike the sst(3) homodimer, the sst(2A)-sst(3) heterodimer did not promote GTPgammaS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst(3)-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst(2A)-sst(3) heterodimer desensitized at a slower rate than the sst(2A) and sst(3) homodimers. Both sst(2A) and sst(3) homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst(2A)-sst(3) heterodimer separated at the plasma membrane, and only sst(2A) but not sst(3) underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst(2A) and sst(3) results in a new receptor with a pharmacological and functional profile resembling that of the sst(2A) receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst(3) receptor function by heterodimerization with sst(2A) or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst(3)-specific binding and signaling in mammalian tissues.     

Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.     

G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions

G protein-coupled receptors are dynamically regulated. Such regulation is frequently associated with covalent posttranslational modifications, such as phosphorylation, and with regulatory elements. G protein-coupled receptor kinases and casein kinase 1alpha play key roles in agonist-dependent receptor phosphorylations. Cross-talk between different receptors frequently involves second messenger-activated proteins, such as protein kinase C and protein kinase A. There is some evidence indicating that such kinases may not only turn off receptors but also switch their coupling to different G proteins. Receptor tyrosine kinases may phosphorylate and regulate G protein-coupled receptors and recent evidence indicates that other kinases, such as Akt/protein kinase B and phosphoinositide 3-kinase, may participate in such regulations as integrators of signalling.Recent approaches have shed new light on G protein-coupled receptor interactions that provide novel mechanisms of action and regulation. G protein-coupled receptor activities go beyond G proteins and receptors can be partners of exquisitely assembled signalling complexes through molecular bridges composed of multidomain proteins. The possibilities of interaction increase enormously through the diversity of structural and functional domains present in complex proteins, many of them just known as predicted sequences.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.     

Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling

Type A and B cholecystokinin (CCK) receptors are highly homologous members of the class-I family of G protein-coupled receptors that bind CCK with high affinity. However, they have divergent structural specificities, with the type A receptor requiring seven carboxyl-terminal residues including a sulfated tyrosine and the type B receptor requiring only the carboxyl-terminal tetrapeptide. The aim of this work was to utilize affinity labeling to determine spatial approximations with photolabile p-benzoyl-l-phenylalanine (Bpa) residues sited at each end of CCK as docked at the type B CCK receptor, contrasting this with analogous work using similar probes docked at the type A receptor. Both probes were fully efficacious, potent agonists that stimulated intracellular calcium in receptor-bearing CHO-CCKBR cells (EC(50) values: Bpa(24) probe, 41 +/- 9 pM; Bpa(33) probe, 15 +/- 3.3 pM). They bound specifically, with high affinity (K(i) values: Bpa(24) probe, 0.60 +/- 0.17 nM; Bpa(33) probe, 0.58 +/- 0.11 nM). Cyanogen bromide cleavage of the covalently labeled receptor suggested the first extracellular loop as the region of labeling by each probe, distinct from the type A CCK receptor regions labeled using the same probes (third loop and amino-terminal tail, respectively). This was confirmed by subsequent enzymatic and chemical cleavage of labeled wild-type and mutant receptors. Sequential cycles of Edman degradation of labeled receptor fragments identified the specific residues within loop one labeled by each probe (Bpa(24) probe labeled Phe(122); Bpa(33) probe labeled Thr(119)). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.     

New roles for beta-arrestins in cell signaling: not just for seven-transmembrane receptors

beta-arrestins, originally discovered as molecules that bind to and desensitize the activated and phosphorylated form of the G protein-coupled beta2-adrenergic receptor (beta2-AR), have recently emerged as multifunctional adaptor/scaffold proteins that dynamically assemble a wide range of multiprotein complexes in response to stimulation of most seven-transmembrane receptors (7TMRs). These complexes mediate receptor signaling, trafficking, and degradation. Moreover, beta-arrestins are increasingly found to perform analogous functions for receptors from structurally diverse classes, including atypical 7TMRs such as frizzled and smoothened, the nicotinic cholinergic receptors, receptor tyrosine kinases, and cytokine receptors, thereby regulating a growing list of cellular processes such as chemotaxis, apoptosis, and metastasis.     

Neurochemical and Molecular Pharmacological Aspects of the GABAB Receptor

Metabotropic gamma-aminobutyric acid (GABA)B receptors are known to modulate the synaptic release of various neurotransmitters in the nervous system. Activation of GABA(B) receptor induces the inhibition of adenylyl cyclase activity, while it does not stimulate the formation of inositol phosphates. Activation of a potassium conductance and suppression of a calcium conductance are also recognized, similarly to some of G protein-coupled receptors. Recent molecular cloning has revealed that GABA(B) receptor possesses a large extracellular domain including the binding site for GABA and seven transmembrane domains. Their molecular structures in the brain are unique and interesting because of heterodimerization consisting of two distinct genes: GABABR1 and GABABR2. Such assembled receptors can be classified as a novel type of the metabotropic receptor superfamily.     

Molecular mechanisms of intercellular communication in the hormonal and neural systems

This paper reviews our studies that have addressed the molecular mechanisms underlying the biosynthesis and reception of extracellular signaling molecules and integrative mechanisms of extracellular-intracellular signaling transmission in biological systems. We introduced recombinant DNA technology into the neuroendocrine system and established the concept that a single peptide precursor encompasses multiple biologically active peptides and brings about coordinate functions in various biological systems. We then developed a novel functional cloning of membrane receptors and ion channels by combining an oocyte expression system with electrophysiology. We molecularly elucidated not only various peptide receptors, including the first demonstration of the molecular entity of a G protein-coupled peptide receptor (GPCR), substance K receptor, and also diverse members of both G protein-coupled metabotropic type and NMDA type of neurotransmitter glutamate receptors. We demonstrated many novel synaptic mechanisms involving distinct types of glutamate receptors in brain function and dysfunction. These include the mechanisms underlying segregation of light-dark signals in visual transmission, discrimination and memory formation in olfactory transmission, and motor co-ordination in the cerebellum, basal ganglia and the retinal network.     

Identifying a Neuromedin U Receptor 2 Splice Variant and Determining Its Roles in the Regulation of Signaling and Tumorigenesis In Vitro

Neuromedin U (NMU) activates two G protein-coupled receptors, NMUR1 and NMUR2; this signaling not only controls many physiological responses but also promotes tumorigenesis in diverse tissues. We recently identified a novel truncated NMUR2 derived by alternative splicing, namely NMUR2S, from human ovarian cancer cDNA. Sequence analysis, cell surface ELISA and immunocytochemical staining using 293T cells indicated that NMUR2S can be expressed well on the cell surface as a six-transmembrane protein. Receptor pull-down and fluorescent resonance energy transfer assays demonstrated that NMUR1, NMUR2 and this newly discovered NMUR2S can not only form homomeric complexes but also heteromeric complexes with each other. Although not activated by NMU itself, functional assay in combination with receptor quantification and radio-ligand binding in 293T cells indicated that NMUR2S does not alter the translocation and stability of NMUR1 or NMUR2, but rather effectively dampens their signaling by blocking their NMU binding capability through receptor heterodimerization. We further demonstrated that NMU signaling is significantly up-regulated in human ovarian cancers, whereas expression of NMUR2S can block endogenous NMU signaling and further lead to suppression of proliferation in SKOV-3 ovarian cancer cells. In contrast, in monocytic THP-1 cells that express comparable levels of NMUR1 and NMUR2S, depletion of NMUR2S restored both the signaling and effect of NMU. Thus, these results not only reveal the presence of previously uncharacterized heteromeric relationships among NMU receptors but also provide NMUR2S as a potential therapeutic target for the future treatment of NMU signaling-mediated cancers.     

Regulation of G protein-coupled receptor endocytosis by ARF6 GTP-binding proteins.

The function of G protein-coupled receptors is regulated by a broad variety of membrane-bound and intracellular proteins. These act in concert to activate signaling pathways that will lead to the desensitization of activated receptors and, for most receptor types, their trafficking to intracellular compartments. This review focuses mainly on the endocytic pathways used by a G protein-coupled receptor and on the proteins that play an essential role in the regulation of the internalization process, most specifically the ADP-ribosylation factors. This family of proteins has been shown to be important for vesicle trafficking between different cellular membranes. The latest findings regarding the molecular mechanisms that regulate internalization of an agonist-stimulated receptor are presented here. Finally, a perspective on how ARF6 proteins might regulate the internalization process is also proposed.