MAGI Proteins Regulate the Trafficking and Signaling of Corticotropin-Releasing Factor Receptor 1 via a Compensatory Mechanism.

Maha M. Hammad,Stephen S. G. Ferguson,Henry A. Dunn

doi:10.5334/1750-2187-11-5

Maha M. Hammad, Stephen S. G. Ferguson + Show 1 more

Open Access

https://doi.org/10.5334/1750-2187-11-5

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Abstract

Corticotropin-releasing factor (CRF) receptor1 (CRFR1) is associated with psychiatric illness and is a proposed target for the treatment of anxiety and depression. Similar to many G protein-coupled receptors (GPCRs), CRFR1 harbors a PDZ (PSD-95/Disc Large/Zona Occludens)-binding motif at the end of its carboxyl-terminal tail. The interactions of PDZ proteins with GPCRs are crucial for the regulation of receptor function. In the present study, we characterize the interaction of all members of the membrane-associated guanylate kinase with inverted orientation PDZ (MAGI) proteins with CRFR1. We show using co-immunoprecipitation that CRFR1 interacts with MAGI-1 and MAGI-3 in human embryonic kidney (HEK293) cells in a PDZ motif-dependent manner. We find that overexpression as well as knockdown of MAGI proteins result in a significant reduction in CRFR1 endocytosis. This effect is dependent on an intact PDZ binding motif for MAGI-2 and MAGI-3 but not MAGI-1. We show that the alteration in expression levels of MAGI-1, MAGI-2 or MAGI-3 can interfere with β-arrestin recruitment to CRFR1. This could explain the effects observed with receptor internalization. We also find that knockdown of endogenous MAGI-1, MAGI-2 or MAGI-3 in HEK293 cells can lead to an enhancement in ERK1/2 signaling but has no effect on cAMP formation. Interestingly, we observe a compensation effect between MAGI-1 and MAGI-3. Taken together, our data suggest that the MAGI proteins, MAGI-1, MAGI-2 and MAGI-3 can regulate β-arrestin-mediated internalization of CRFR1 as well as its signaling and that there is a compensatory mechanism involved in regulating the function of the MAGI subfamily.

Highlights

Membrane-associated guanylate kinase (MAGUK) family proteins are synaptic scaffolding proteins within a structured protein network responsible for the spatial organization of the presynaptic and postsynaptic compartments
membrane-associated guanylate kinase with inverted orientation postsynaptic density protein 95 (PSD-95)/Disc Large/Zona Occludens (PDZ) (MAGI) proteins interact with corticotropin-releasing factor receptor 1 (CRFR1) via the PDZ-binding motif independent of Corticotropin-releasing factor (CRF) activation The different members of the MAGI subfamily of proteins were previously shown to interact with different G protein-coupled receptor (GPCR)
We found that hexahistidine tagged (His)-MAGI-1 and His-MAGI-3 co-immunoprecipitated with HA-CRFR1 but not CRFR1-DTAV (Fig. 1A and 1C) and that the interaction with HA-CRFR1 was not altered following agonist treatment with 100 nM CRF for 30 min (Fig. 1B and 1D)

Summary

Introduction

Membrane-associated guanylate kinase (MAGUK) family proteins are synaptic scaffolding proteins within a structured protein network responsible for the spatial organization of the presynaptic and postsynaptic compartments They play a crucial role in the formation and function of synapses in the central nervous system (CNS) by regulating multiple aspects of synapse physiology such as synaptogenesis, receptor trafficking, synaptic function, and plasticity [1,2,3]. MAGUKs are well-conserved throughout evolution and are widely expressed in the brain and periphery They include multiple subfamilies including membrane palmitoylated proteins (MPPs), zona occludens (ZO), caspase recruitment domain-containing MAGUK protein (CARMA), discs large (DLGs) and MAGUK with inverted orientation PSD-95/Disc Large/Zona Occludens (PDZ) (MAGI) proteins [4]. One of the important subfamilies of MAGUKs is the membrane-associated guanylate kinase with inverted

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