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.
Laboratoire de Génomique Fonctionnelle, CNRS UPR2580, CCIPE, 141 rue de la Cardonille, 34094 Montpellier CEDEX 05, France.
Lara
L
Joubert
Brendon
B
Hanson
Gaël
G
Barthet
Michèle
M
Sebben
Sylvie
S
Claeysen
Wanjin
W
Hong
Philippe
P
Marin
Aline
A
Dumuis
Joël
J
Bockaert
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