New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting.
The 5-hydroxytryptamine type 4 receptor (5-HT4R) is involved in learning, feeding, respiratory control and gastrointestinal transit. This receptor is one of the G-protein-coupled receptors for which alternative mRNA splicing generates the most variants that differ in their C-terminal extremities. Some 5-HT4R variants (a, e and f) express canonical PDZ ligands at their C-termini. Here, we have examined whether some mouse 5-HT4R variants associate with specific sets of proteins, using a proteomic approach based on peptide-affinity chromatography, two-dimensional electrophoresis and mass spectrometry. We have identified ten proteins that interact specifically with the 5-HT4(a)R and three that only associate with the 5-HT4(e)R. Most of them are PDZ proteins. Among the proteins that associated specifically with the 5-HT4(a)R variant, NHERF greatly modified its subcellular localization. Moreover, NHERF recruited the 5-HT4(a)R to microvilli, where it localized with activated ezrin, consistent with the role of 5-HT4(a)R in cytoskeleton remodelling. The 5-HT4(a)R also interacted with both the constitutive and inducible (upon methamphetamine treatment) forms of the recently cloned sorting nexin 27 (SNX27a and b, respectively). We found that SNX27a redirected part of 5-HT4(a)R to early endosomes. The interaction of the 5-HT4R splice variants with distinct sets of PDZ proteins might specify their cellular localization as well as their signal transduction properties.
Joubert
Lara
L
Laboratoire de Génomique Fonctionnelle, CNRS UPR2580, CCIPE, 141 rue de la Cardonille, 34094 Montpellier CEDEX 05, France.
Hanson
Brendon
B
Barthet
Gaël
G
Sebben
Michèle
M
Claeysen
Sylvie
S
Hong
Wanjin
W
Marin
Philippe
P
Dumuis
Aline
A
Bockaert
Joël
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D002845:Chromatography; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008694:Methamphetamine / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009474:Neurons / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics; D044422:Receptors, Serotonin, 5-HT4 / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers; D058305:Sorting Nexins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D033921:Vesicular Transport Proteins; D000099149:Ezrin
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008694:Methamphetamine; D010455:Peptides; D010750:Phosphoproteins; D044422:Receptors, Serotonin, 5-HT4; D017923:Sodium-Hydrogen Exchangers; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; D000099149:Ezrin; C500393:Snx27 protein, mouse; C093376:sodium-hydrogen exchanger regulatory factor