CRHR1-WT and CRHR1-STAVA localize throughout the neuronal plasma membrane including the excitatory post synapse.

<p>Primary hippocampal neurons at DIV 14–20 were transduced with CRHR1-WT and CRHR1-STAVA using AAV8. (A) The spatial pattern of CRHR1-WT and CRHR1-STAVA expression visualized by immunostaining of the GFP tag was confirmed using an anti-CRHR1 antibody. (B) Microtubule-associated protein 2 (MAP2) staining revealed the dendritic presence of the receptor. (C) Co-staining with the axon initial segment marker ankyrin G demonstrated CRHR1-WT and CRHR1-STAVA localization within axons. (D) The presynaptic marker synapsin indicated that CRHR1-WT and CRHR1-STAVA are present in the adjacent post synapse but not in the presynaptic axon terminal. (E) CRHR1-WT and CRHR1-STAVA did not co-localize with the inhibitory postsynaptic marker gephyrin but (F) they co-localized with the MAGUK and excitatory postsynaptic marker PSD95 in spines. (G) CRHR1-WT and mutant co-localized with the candidate interaction partner SAP97.</p

CRHR1 and candidate MAGUKs are co-expressed in the adult mouse brain.

<p>(A–F) Expression of mRNA of CRHR1, PSD95, SAP97, SAP102, PSD93, and MAGI2 was assessed by single <i>in situ</i> hybridization on consecutive coronal brain sections of wild-type mice. For comparison, representative photomicrographs of autoradiographs are shown in a false color display. CRHR1 expression is displayed in green, and PSD95, SAP97, SAP102, PSD93, and MAGI2 expression are displayed in red. The yellow signal reveals brain structures of overlapping expression of CRHR1 with respective MAGUKs. (G–K) Double <i>in situ</i> hybridization revealed co-expression of CRHR1 and interacting partners at the cellular level. Depicted are representative photomicrographs of coronal brain sections of CRHR1-GFP reporter mice. Blue boxes depict individual neurons shown below as magnifications (left: single MAGUK positive neuron, right: MAGUK and CRHR1 double positive neuron). Arrows indicate single positive cells stained by silver grains for CRHR1, and red arrowheads indicate cells expressing the candidate MAGUK only. White arrowheads indicate double-positive cells co-expressing CRHR1 and its respective interaction partners. CA: cornu ammonis, RTN: reticular thalamic nucleus.</p

CRHR1 PDZ binding motif is required for clustering with interacting MAGUKs.

<p>CRHR1-WT (A), CRHR1-STAVA (G), or the interacting proteins (first row) were transiently transfected alone. (B–F) Wild-type CRHR1 or (H–L) CRHR1-STAVA were transiently transfected in HEK293 together with (B, H) PSD95-flag, (C, I) HA-SAP97, (D, J) flag-SAP102, (E, K) PSD93-GFP and (F, L) myc-MAGI2. CRHR1-WT co-transfection with the respective interacting partner resulted in a co-clustering of both proteins (B–F). (H–L) Co-transfection of CRHR1-STAVA with the respective interacting partner did not result in any clustering, but both proteins appeared with a subcellular distribution similar to the individual transfections. Immunostaining was performed against the HA tag of HA-CRHR1 or HA-CRHR1-STAVA when co-transfected with PSD95-flag or flag-SAP102. Immunostaining was performed against the flag tag of flag-CRHR1 or flag-CRHR1-STAVA when co-transfected with PSD93-GFP, HA-SAP97, or myc-MAGI2 respectively.</p

CRHR1 interaction with MAGI2 depends on the PDZ binding motif.

<p>Co-IPs were performed using lysates of HEK293 cells transiently transfected as indicated. (A) flag-CRHR1 but not flag-CRHR1-STAVA interacted with myc-MAGI2, and myc-MAGI2 was co-immunoprecipitated with flag-CRHR1 but not with flag-CRHR1-STAVA. (B) flag-CRHR1 was co-immunoprecipitated with myc-MAGI2, myc-MAGI2 WW + PDZ1, and myc-MAGI2 PDZ2-5 but not with myc-MAGI2 PDZ0 + GuK. Co-IPs were also successful in the other direction. The bands for the mutants of MAGI2 in the control blot are highlighted when necessary (→). (C) flag-CRHR1 neither interacted with syntenin-1-myc under basal conditions, nor after CRH (100 nM) treatment. Similarly syntenin-1-myc was not co-immunoprecipitated with flag-CRHR1. CRHR1 showed high molecular weight complexes (*) together with the momomeric form (>). Dashed lines indicate that the samples were run on the same immunoblot (IB), however, not in adjacent lanes. Continuous lines separate different IBs from the same experiment. The ~55 kDa band in the IB of anti-myc represents the heavy chain of the primary antibody. IP: immunoprecipitation.</p

Summary of interactions of CRHR1 with MAGUKs, as revealed by co-immunoprecipitation (Co-IP).

<p>+ interaction, = comparable Co-IP efficiency, > Co-IP more efficient when the immunoprecipitation (IP) was done against CRHR1, < Co-IP more efficient when the IP was done against interactor,—no interaction, n.a. not analyzed.</p

CRHR1 interaction with SAP97, SAP102 and PSD93 depends on the PDZ binding motif.

<p>Co-IPs were performed using lysates of HEK293 cells transiently transfected as indicated. (A) myc-GFP-CRHR1 but not myc-GFP-CRHR1STAVA was co-immunoprecipitated with HA-SAP97, and similarly, HA-SAP97 was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. (B) HA-SAP97 PDZ1-3 and HA-SAP97 PDZ1-2 were co-immunoprecipitated with myc-GFP-CRHR1 and accordingly, these SAP97 variants were co-immunoprecipitated with myc-GFP-CRHR1 using an anti-myc antibody in the Co-IP. No interaction was observed for HA-SAP97 PDZ1. HA-SAP97 PDZ1-2 detected by the anti-HA antibody following the anti-myc Co-IP has the same molecular weight and thus is indistinguishable from the heavy chain of the anti-myc antibody (→). (C) myc-GFP-CRHR1 but not myc-GFP-CRHR1-STAVA was co-immunoprecipitated with flag-SAP102. Similarly, flag-SAP102 was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. Moreover, flag-SAP102 PDZ3 was not detected following an immunoprecipitation (IP) of myc-GFP-CRHR1. (D) flag-CRHR1 but not flag-CRHR1-STAVA was co-immunoprecipitated with GFP-PSD93 and accordingly, GFP-PSD93 was co-immunoprecipitated with flag-CRHR1 but not with flag-CRHR1-STAVA. CRHR1 always showed high molecular weight complexes (*) together with the monomeric form (>). Therefore, high molecular weight complexes are also shown when necessary. Dashed lines indicate that the samples were run on the same immunoblot (IB), however, not in adjacent lanes. Continuous lines separate different IBs from the same experiment. (B) The ~55 kDa band represents the heavy chain of the primary antibody.</p