Doctor of Philosophy
Dr. Stephen S.G. Ferguson
Previous studies identified a crosstalk mechanism whereby CRFR1 sensitized 5-HT2AR-mediated signaling via interactions with PDZ domain-containing proteins: a mechanism that may underlie stress-induced anxiety and depression. This prompted an investigation into uncovering which PDZ domain-containing proteins could regulate the crosstalk between these two receptors, and how they could be regulated individually. In the current studies, a subset of PDZ domain-containing proteins were identified that may interact with CRFR1 and 5-HT2AR. The focus narrowed to two candidates previously implicated in psychiatric disease: SAP97 and PSD-95. We confirmed SAP97 and PSD-95 as interacting partners of CRFR1 in adult mouse cortex via co-immunoprecipitation. Both proteins exhibited functional regulation of CRFR1 by antagonizing CRFR1 endocytosis in HEK293 cells, measured by flow cytometry. Additionally, PSD-95 suppressed β-arrestin2 recruitment, thereby providing a potential mechanism for antagonizing endocytosis. Although neither SAP97 nor PSD-95 appeared to play a significant role in CRFR1-mediated cAMP signaling, endogenous SAP97 was integral for CRF-mediated ERK1/2 phosphorylation in HEK293 and AtT20 cells. Despite extensive sequence homology between SAP97 and PSD-95, PSD-95 did not appear to play a significant role in CRF-mediated ERK1/2 phosphorylation. Thus, we begin to understand subtle signaling biases between these two proteins. As PSD-95 was already documented to regulate 5-HT2AR, we investigated if SAP97 could play a role in regulating 5-HT2AR function. The interaction between SAP97 and 5-HT2AR was confirmed in adult mouse cortex. As was seen with CRFR1, SAP97 antagonized 5-HT2AR endocytosis. Although SAP97 did not appear to significantly modulate Gs-coupled signaling via CRFR1, the endogenous expression of SAP97 was integral for maximal Gq-coupled signaling via 5-HT2AR. Endogenous SAP97 was also required for ERK1/2 phosphorylation, and this regulatory role appears to be downstream of receptor interactions. Finally, we were unable to prevent the CRFR1-mediated sensitization of 5-HT2AR-mediated signaling by knocking down either SAP97 or PSD-95 using shRNA. Therefore, neither SAP97 nor PSD-95 appear to be exclusively involved in this heterologous crosstalk mechanism. Nevertheless, we have identified SAP97 and PSD-95 as novel regulators of CRFR1 function, and SAP97 as a novel regulator of 5-HT2AR function. These functional interactions may be targeted for the treatment of CRFR1- and 5-HT2AR-mediated mood disorders.
Dunn, Henry A., "Regulation of CRFR1 and 5-HT2AR by PDZ Domain-Containing Proteins SAP97 and PSD-95" (2014). Electronic Thesis and Dissertation Repository. 2585.