Fig 1.
CRHR1 and candidate MAGUKs are co-expressed in the adult mouse brain.
(A–F) Expression of mRNA of CRHR1, PSD95, SAP97, SAP102, PSD93, and MAGI2 was assessed by single in situ 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 in situ 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.
Fig 2.
Expression of CRHR1 and its interacting MAGUKs in primary hippocampal neurons.
The mRNA levels of CRHR1, its ligand CRH, the related receptor CRHR2 and the candidate partners PSD95, SAP97, SAP102, PSD93, and MAGI2 were determined by quantitative real time PCR and normalized to ribosomal protein L19 (RPL-19) mRNA expression. Expression levels were analyzed at DIV 0 (days in vitro) and DIV 21. n.d.: not detected.
Fig 3.
PDZ binding motif-mediated interaction of CRHR1 with PSD95 depends on PDZ1 and PDZ2.
Co-immunoprecipitations (Co-IPs) were performed using lysates of HEK293 cells transiently transfected as indicated. (A) PSD95 PDZ1-3-flag was co-immunoprecipitated with wild-type myc-CRHR1-STAV and to variable degrees with myc-CRHR1-STAA, myc-CRHR1-SAAV, and myc-CRHR1-ATAV but not with the myc-CRHR1-STAVA mutant. (B) PSD95-flag was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. Accordingly, myc-GFP-CRHR1, but not myc-GFP-CRHR1-STAVA, was co-immunoprecipitated with PSD95. PSD95-flag was not co-immunoprecipitated with the phospho-mimicking variant myc-GFP-CRHR1-SEAV, but with the phospho-mimicking variant myc-GFP-CRHR1-ETAV. Accordingly, myc-GFP-CRHR1-SEAV was not co-immunoprecipitated with PSD95-flag, but myc-GFP-CRHR1-ETAV was co-immunoprecipitated with PSD95-flag. (C) myc-GFP-CRHR1 was co-immunoprecipitated with the PSD95 mutants PSD95 PDZ1-flag and PSD95 PDZ2-3-flag, but not with PSD95Δ PDZ1-3-flag or PSD95 PDZ3-flag. Complementary results were obtained when the Co-IP was performed against myc-GFP-CRHR1. In lanes 8 and 10 a slight cross-reactivity with PSD95 PDZ2-3-flag (→)—appearing as a band below myc-GFP-CRHR1—was observed. 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 anti-flag IB represents the heavy chain of the primary antibody. IP: immunoprecipitation.
Fig 4.
CRHR1 interaction with SAP97, SAP102 and PSD93 depends on the PDZ binding motif.
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.
Fig 5.
CRHR1 interaction with MAGI2 depends on the PDZ binding motif.
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.
Fig 6.
Summary of interactions of CRHR1 with MAGUKs, as revealed by co-immunoprecipitation (Co-IP).
+ 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.
Fig 7.
CRHR1-WT and CRHR1-STAVA localize throughout the neuronal plasma membrane including the excitatory post synapse.
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.
Fig 8.
CRHR1 PDZ binding motif is required for clustering with interacting MAGUKs.
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.