Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

Abstract

Cholesterol is an essential constituent of cell membranes. Recently, the discovery of cholesterol recognition amino acid consensus (CRAC) on proteins indicated a putative direct, non-covalent interaction between cholesterol and proteins. In the present study, we evaluated the presence of a CRAC motif and its inverted version (CARC) in the transmembrane region (TMR) of the tyrosine kinase receptor family (RTK) in several species using in silico methods. CRAC motifs were found across all species analyzed, while CARC was found only in vertebrates. The tropomyosin-related kinase B (TRKB), a member of the RTK family, is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon recognition of the conserved CARC motif in the TRKB, we compared the effect of point mutations in CARC on structural changes in the TMR of mouse TRKB. The alignment of wild-type and mutant TMR indicates small morphological changes across the 6 mutations analyzed (Y433F, Y433C, Y433A, V437K, R427A, and the double mutation R427A/Y433F), as demonstrated by the root-mean-squared deviation values for the superimposed structures. A molecular dynamics simulation with the mouse TRKB TMR sequence indicated that cholesterol interaction with the TRKB CARC motif is reduced by the R427A/Y433F mutation. Experimental data assayed by fluorescence recovery after photobleaching indicated a reduction in brain-derived neurotrophic factor-induced mobility of TRKB.R427A/Y433F in the spine of cultured hippocampal neurons. Therefore, CARC/CRAC motifs may have a role in the function of the RTK family TMR.

Chondroitinase and Antidepressants Promote Plasticity by Releasing TRKB from Dephosphorylating Control of PTPσ in Parvalbumin Neurons

“…For the experiments assessing the effect of PTPσ genetic deficiency on pTRKB in vitro, cortical cell cultures were prepared from balb/c mouse embryos (PTPσ -/-, PTPσ +/and their WT littermates) extracted on embryonic day 18 (E18) using the same protocol as described above for the plasmid purchased from OriGene (#RR209636), pCMV6-Entry vector with C-terminal Myc-DDK Tag (#PS100001) was used to transfect control cells. HEK293T were transfected with GFP-tagged fulllength TRKB plasmid (HP220GFP-TRKB) (Haapasalo et al, 2001) or TRKB carrying Y433F/R427A mutation (Cannarozzo et al, 2020).…”

“…MG87.TRKA and MG87.TRKB cells were transfected with PTPσ (RefSeq number NM_019140) Myc-DDK-tagged open-reading frame (ORF) plasmid purchased from OriGene (#RR209636), pCMV6-Entry vector with C-terminal Myc-DDK Tag (#PS100001) was used to transfect control cells. HEK293T were transfected with GFP-tagged full-length TRKB plasmid (HP220GFP-TRKB; Haapasalo et al, 2001 ) or TRKB carrying Y433F/R427A mutation ( Cannarozzo et al, 2020 ).…”

“…Therefore, we asked whether disruption in TRKB:PTPσ interaction after fluoxetine treatment is potentially mediated by the antidepressant's binding to the same region of TRKB where interaction with PTPσ takes place. We transfected HEK293T cells with either a wild-type TRKB plasmid or a TRKB plasmid carrying two point mutations in the transmembrane region of TRKB (arginine R427 mutated to alanine, R427A; and tyrosine Y433 mutated to phenylalanine, Y433F); these point mutations disrupt a cholesterol interaction site in the TRKB transmembrane region critical for antidepressant interaction (Cannarozzo et al, 2020). R427A/Y433F mutation caused a dramatic decrease in TRKB:PTPσ interaction levels, however, the interaction was not completely lost, suggesting that multiple sites of interaction may exist.…”

Chondroitinase and antidepressants promote plasticity by releasing TRKB from dephosphorylating control of PTPσ in parvalbumin neurons

“…MG87.TRKA and MG87.TRKB cells were transfected with PTPσ (RefSeq number NM_019140) Myc-DDK-tagged open-reading frame (ORF) plasmid purchased from OriGene (#RR209636), pCMV6-Entry vector with C-terminal Myc-DDK Tag (#PS100001) was used to transfect control cells. HEK293T were transfected with GFP-tagged full-length TRKB plasmid (HP220GFP-TRKB)(Haapasalo et al, 2001) or TRKB carrying Y433F/R427A mutation (Cannarozzo et al, 2020).…”

Chaperone Sigma1R and Antidepressant Effect

“…The mechanism of regulation of BDNF-dependent processes may be associated with the ability of Sigma1R to bind and transport cholesterol during ligand activation due to the presence of cholesterol recognition or interaction of amino acid consensus sequences (CRAC/CARC) [ 66 , 73 , 167 ]. Pilot studies have shown that CRAC/CARC motifs are present in the trkB sequence [ 168 ], and cholesterol itself is able to stimulate trkB phosphorylation [ 169 ]. The activity of both Sigma1R and trkB is associated with their localization in cholesterol-rich lipid domains [ 73 , 170 , 171 , 172 , 173 ], while Sigma1R ligands fluoxetine and escitalopram have the ability to accumulate in lipid rafts [ 174 ].…”