Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif.

Julia Bender,Martin Biel,Stylianos Michalakis,Wolfgang Wurst,Thor C Møller,Johannes Breu,Maik Engeholm,Karen L Martinez,Jan M Deussing,Erich E Wanker,Tamas Rasko,Marion S Ederer

doi:10.1371/journal.pone.0136768

Abstract

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.

Highlights

The corticotropin-releasing hormone receptor type 1 (CRHR1) is an important regulator of neuroendocrine, behavioral, and autonomic response to stress [1]
As the most abundant candidate interaction partners, we identified members of the membrane-associated guanylate kinase (MAGUK) family: PSD95, synapse-associated protein 97 (SAP97), SAP102, and MAGI2
As a first step toward the validation of the identified interactions we assessed whether CRHR1 and the potential interaction partners are co-expressed in the mouse brain which would be a prerequisite for a direct physical interaction

Summary

Introduction

The corticotropin-releasing hormone receptor type 1 (CRHR1) is an important regulator of neuroendocrine, behavioral, and autonomic response to stress [1]. The conditional inactivation of CRHR1 within the murine forebrain demonstrated that limbic CRHR1 signaling modulates anxiety-related behavior, independent of its role in the neuroendocrine stress response via the hypothalamic–pituitary– adrenocortical axis [4]. CRHR1 is a G protein-coupled receptor (GPCR) of the B1 family and preferentially signals via Gsα, resulting in the activation of the adenylyl cyclase/protein kinase A pathway [6, 7]. Up to now CRHR1 interactions with G proteins, arrestins, and G protein-coupled receptor kinases have extensively been studied [13, 14], whereas its interactions with other accessory or GPCR-interacting proteins, which would provide further specificity to CRHR1 signaling or determine the activation of particular downstream pathways, are largely unknown

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Conclusion