Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif.
The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.
Max Planck Institute of Psychiatry, Department of Stress Neurobiology and Neurogenetics, Molecular Neurogenetics, Munich, Germany.
Julia
J
Bender
Max Planck Institute of Psychiatry, Munich, Germany.
Maik
M
Engeholm
Max Planck Institute of Psychiatry, Department of Stress Neurobiology and Neurogenetics, Molecular Neurogenetics, Munich, Germany.
Marion S
MS
Ederer
Max Planck Institute of Psychiatry, Munich, Germany.
Johannes
J
Breu
University of Copenhagen, Department of Chemistry & Nano-Science Center, Copenhagen, Denmark.
Thor C
TC
Møller
Center for Integrated Protein Science Munich (CIPSM) and Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.
Stylianos
S
Michalakis
Max Delbrueck Center for Molecular Medicine, Berlin-Buch, Germany.
Tamas
T
Rasko
Max Delbrueck Center for Molecular Medicine, Berlin-Buch, Germany.
Erich E
EE
Wanker
Center for Integrated Protein Science Munich (CIPSM) and Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.
Martin
M
Biel
University of Copenhagen, Department of Chemistry & Nano-Science Center, Copenhagen, Denmark.
Karen L
KL
Martinez
Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany; German Center for Neurodegenerative Diseases within the Helmholtz Association, Munich, Germany; Technische Universität München-Weihenstephan, Lehrstuhl für Entwicklungsgenetik c/o Helmholtz Zentrum München, Neuherberg, Germany.
Wolfgang
W
Wurst
Max Planck Institute of Psychiatry, Department of Stress Neurobiology and Neurogenetics, Molecular Neurogenetics, Munich, Germany.
Jan M
JM
Deussing
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D051528:Guanylate Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D054731:PDZ Domains; D010766:Phosphorylation; D011485:Protein Binding; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism; D000099052:CRF Receptor, Type 1
D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1; D051528:Guanylate Kinases