The Third Intracellular Loop of the 5‐Hydroxytryptamine<sub>2A</sub> Receptor Determines Effector Coupling Specificity

Oksenberg, Donna; Havlik, Sona; Peroutka, Stephen J.; Ashkenazi, Avi

doi:10.1046/j.1471-4159.1995.64041440.x

Cited by 18 publications

(8 citation statements)

References 20 publications

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“…This is an intriguing result that suggests there are additional ciliary localization sequences within Sstr3 and Htr6. The i3 loops of GPCRs, including somatostatin and serotonin receptors, are normally associated with G protein coupling and desensitization (Oksenberg et al, 1995;Gelber et al, 1999;Hipkin et al, 2000). This is the first time the i3 loop has been implicated in GPCR ciliary localization.…”

Section: Discussionmentioning

confidence: 93%

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Berbari¹,

Johnson²,

Lewis³

et al. 2008

MBoC

335

355

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Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight. INTRODUCTIONPrimary cilia are appendages that project from almost all human cell types (Wheatley et al., 1996). It is generally accepted that primary cilia serve important specialized signaling functions (Pazour and Witman, 2003;Marshall and Nonaka, 2006;Singla and Reiter, 2006). In the eye, photoreceptors, which are modified primary cilia, sense and respond to light. In the nose, specialized olfactory cilia detect odors and initiate signaling cascades in olfactory neurons. In the kidney, it is proposed that bending of cilia on epithelial cells by fluid flow triggers an increase in intracellular calcium mediated by an ion channel located on the cilium (Praetorius and Spring, 2001;Nauli et al., 2003). In each case, the function of the cilium is defined by the signaling proteins that are enriched in the ciliary membrane (i.e., light receptors, odorant receptors, and mechanoreceptors). Importantly, disruption of the signaling mediated by these receptors can cause disease and altered development (Davenport and Yoder, 2005;Hildebrandt and Otto, 2005;Pan et al., 2005;Bisgrove and Yost, 2006). Yet, the specific signaling proteins that localize to the vast majority of cilia in the mammalian body are unknown. Thus, the functions of primary cilia on most cell types in the body are unknown.Neuronal primary cilia are abundant throughout the rodent brain (Bishop et al., 2007). The functional importance of these cilia is suggested by the fact that several human ciliary disorders, including Bardet-Biedl syndrome (BBS), Joubert syndrome (JS), and Meckel syndrome (MKS), have prominent functional and structural CNS phenotypes (Badano et al., 2006). Although the specific functions of neuron...

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Section: Discussionmentioning

confidence: 93%

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Berbari¹,

Johnson²,

Lewis³

et al. 2008

MBoC

335

355

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“…These residues reside within transmembrane domain 6 (TM6), which is directly coupled to the third intracellular loop (IL3), between TM5 and TM6. This loop is implicated as being important for the ability of the 5-HT 2A receptor (and all related GPCRs) to interact with and activate the appropriate G proteins and thus affect second messenger production, particularly G␣ q activation of PI hydrolysis (Kubo et al, 1988;Wess et al, 1989Wess et al, , 1990Oksenberg et al, 1995;Hill-Eubanks et al, 1996). It would seem that agonist or antagonist character, affinity, and potency of a particular ligand is dependent on the ability of the ligand to interact with specific TM6 residues and induce or suppress movement of this helix.…”

Section: Discussionmentioning

confidence: 99%

Molecular Interaction of Serotonin 5-HT_2AReceptor Residues Phe339^(6.51)and Phe340^(6.52)with SuperpotentN-Benzyl Phenethylamine Agonists

Braden

Parrish²,

Naylor³

et al. 2006

Mol Pharmacol

202

204

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Experiments were conducted to examine the molecular basis for the high affinity and potency of a new class of 5-HT 2A receptor agonists, N-benzyl phenethylamines. Competition binding assays at several serotonin receptors confirmed that an N-arylmethyl substitution was necessary for affinity increases up to 300-fold over simple N-alkyl homologs, as well as enhanced selectivity for 5-HT 2A versus 5-HT 2C and 5-HT 1A receptors. PI hydrolysis functional assays confirmed that these Nbenzyl phenethylamines are potent and highly efficacious agonists at the rat 5-HT 2A receptor. Virtual docking of these compounds into a human 5-HT 2A receptor homology model indicated that the N-benzyl moiety might be interacting with Phe339 (6.51) , whereas the phenethylamine portion was likely to be interacting with Phe340 (6.52) . Experiments in h5-HT 2A receptors with Phe339 (6.51) L and Phe340 (6.52) L mutations seem to support this hypothesis. Dramatic detrimental effects on affinity, potency, and intrinsic activity were observed with the Phe339 (6.51) L mutation for all N-benzyl analogs, whereas most N-unsubstituted phenethylamines and traditional agonists were only weakly affected, if at all. Consistent with other published studies, the Phe340 (6.52) L mutation detrimentally affected affinity, potency, and intrinsic activity of nearly all compounds tested, although a strong change in intrinsic activity was not seen with most N-aryl analogs. These data further validate the topology of our h5-HT 2A receptor homology model. It is noteworthy that this study is the first to identify a hitherto unrecognized role for residue 6.51 in agonist activation of a serotonin G protein-coupled receptor (GPCR), whereas most previous reports have suggested a varied and sometimes contradictory role in homologous GPCRs.Agonist activity at the serotonin 2A (5-HT 2A ) receptor is essential for the psychopharmacology of serotonergic psychedelics such as LSD, DOI, psilocin, and 5-MeO-DMT, compounds with unique and dramatic effects on certain aspects of consciousness (Nichols 2004). Moreover, we have recently identified a functionally selective 5-HT 2A receptor agonist that selectively activates phosphoinositide turnover over production of eicosanoids (McLean et al., 2006a). A key aspect to understanding the effects on consciousness of psychedelics is the study of the receptor-ligand interaction at the molecular level and how it modulates second messenger generation subsequent to receptor activation.We have been particularly interested in experimental val-

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“…These findings agree with several studies suggesting that amphipathic ␣-helices are involved in determining receptor-G protein binding (60,61) and may, in fact, be intrinsic to G␣ q coupling as suggested by the recent structure of G␣ q -coupled squid rhodopsin (62). Furthermore, merely introducing the i3 loop of the G␣ q -coupled 5-HT 2A receptor into the G␣ i -coupled 5-HT 1B receptor (5-HT 1B/2A ) is sufficient to shift its coupling specificity from inhibition of adenylyl cyclase to phospholipase C activation (63). Together with previous studies showing that introduction of phosphorylated residues into the cytoplasmic domains of many GPCRs inhibits signaling, it seems likely that Ser-314 phosphorylation functionally uncouples the 5-HT 2A receptor from its cognate G protein.…”

Section: Rsk2 Phosphorylates the 5-ht 2a Receptor I3mentioning

confidence: 99%

Ribosomal S6 Kinase 2 Directly Phosphorylates the 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptor, Thereby Modulating 5-HT2A Signaling

Strachan

Sheffler

Willard

et al. 2009

Journal of Biological Chemistry

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The 5 We report here that RSK2 directly phosphorylates the 5-HT 2A receptor i3 loop at the conserved residue Ser-314, thereby modulating 5-HT 2A receptor signaling. Furthermore, these studies led to the discovery that RSK2 is required for epidermal growth factor-mediated heterologous desensitization of the 5-HT 2A receptor. We arrived at these conclusions via multiple lines of evidence, including in vitro kinase experiments, tandem mass spectrometry, and site-directed mutagenesis. Our findings were further validated using phospho-specific Western blot analysis, metabolic labeling studies, and whole-cell signaling experiments. These results support a novel regulatory mechanism in which a downstream effector of the ERK/MAPK pathway directly interacts with, phosphorylates, and modulates signaling of the 5-HT 2A serotonin receptor. To our knowledge, these findings are the first to demonstrate that a downstream member of the ERK/MAPK cascade phosphorylates a GPCR as well as mediates cross-talk between a growth factor and a GPCR.The 5-HT 2A 2 receptor plays a key role in transducing a variety of cellular signals elicited by 5-HT in both peripheral and central tissues (75). These include the following: 1) platelet aggregation (1); 2) vascular and nonvascular smooth muscle contraction (2); 3) cognitive processes underlying working memory (3); 4) modulating sensory processing in the cortex (4); and 5) mediating the actions of most, but not all, hallucinogens that act as 5-HT 2A receptor agonists (5, 6). Moreover, dysregulation of the 5-HT 2A receptor has been linked to the etiology of several psychiatric disorders, including depression, anxiety, and schizophrenia, thus highlighting the importance of gaining a more thorough understanding of the precise regulation of 5-HT 2A receptors (7).The 5-HT 2A receptor belongs to the GPCR superfamily that encompasses molecular targets for an extreme diversity of endogenous and exogenous ligands that are essential for nearly every physiological process (8). Extensive studies focusing on the G protein-coupled receptor kinase-arrestin pathway and the second messenger-dependent protein kinase (cAMPdependent protein kinase and protein kinase C (PKC)) pathways suggest that direct GPCR phosphorylation remains the predominant mechanism for rapidly attenuating the signaling of many GPCRs (9, 10). Additional kinases have also been shown to phosphorylate GPCRs, and it is likely that many yet to be discovered kinases regulate GPCR signaling (11).Several studies have demonstrated that PKC modulates 5-HT 2A receptor signaling in vivo and in vitro. Our early studies (12) showed that activation of PKC by phorbol dibutyrate inhibited 5-HT 2A -mediated signaling. Many subsequent studies in a variety of cellular contexts have replicated these observations (13-18). In addition to PKC, recent reports suggest that calmodulin-dependent protein kinase II and G protein-coupled receptor kinase 2/3 regulate 5-HT 2A signaling (18,19), although the role of G protein-coupled receptor kinases is cellspecific (20)...

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The Third Intracellular Loop of the 5‐Hydroxytryptamine_2A Receptor Determines Effector Coupling Specificity

Cited by 18 publications

References 20 publications

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Molecular Interaction of Serotonin 5-HT_2AReceptor Residues Phe339^(6.51)and Phe340^(6.52)with SuperpotentN-Benzyl Phenethylamine Agonists

Ribosomal S6 Kinase 2 Directly Phosphorylates the 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptor, Thereby Modulating 5-HT2A Signaling

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The Third Intracellular Loop of the 5‐Hydroxytryptamine2A Receptor Determines Effector Coupling Specificity

Cited by 18 publications

References 20 publications

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Molecular Interaction of Serotonin 5-HT2AReceptor Residues Phe339(6.51)and Phe340(6.52)with SuperpotentN-Benzyl Phenethylamine Agonists

Ribosomal S6 Kinase 2 Directly Phosphorylates the 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptor, Thereby Modulating 5-HT2A Signaling

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The Third Intracellular Loop of the 5‐Hydroxytryptamine_2A Receptor Determines Effector Coupling Specificity

Molecular Interaction of Serotonin 5-HT_2AReceptor Residues Phe339^(6.51)and Phe340^(6.52)with SuperpotentN-Benzyl Phenethylamine Agonists