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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,
WWand 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.
