Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

Abstract

Protein homeostasis (proteostasis) deficiency is recognized as a contributing factor to many neurodegenerative, neurological, and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is not completely understood. In this investigation, we focus on characterizing the biogenesis pathway of a multi-subunit neuroreceptor, the gamma-aminobutyric acid type A (GABAA) receptor. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched GABAA receptor-interacting chaperone. Here, we show that Hsp47 enhances neuronal GABAA receptor functional surface expression, acting after Binding immunoglobulin Protein (BiP), to preferentially bind the folded conformation of GABAA receptors. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. These Hsp47 properties are also extended to other Cys-loop receptors, including nicotinic acetylcholine receptors. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 also plays a critical and general role in the maturation of multi-subunit neuroreceptors.

Pharmacological chaperones restore proteostasis of epilepsy-associated GABAAreceptor variants

“…For FRET experiments on GABA A receptors, (1) for FRET pair samples, HEK293T cells on coverslips were transfected with α1-CFP (donor) (0.5 μg), β2-YFP (acceptor) (0.5 μg), and γ2 (0.5 μg) subunits; (2) for the donor-only samples, to determine the spectral bleed-through (SBT) parameter for the donor, HEK293T cells were transfected with α1-CFP (donor) (0.5 μg), β2 (0.5 μg), and γ2 (0.5 μg) subunits; (3) for the acceptor-only samples, to determine the SBT parameter for the acceptor, HEK293T cells were transfected with α1 (0.5 μg), β2-YFP (acceptor) (0.5 μg), and γ2 (0.5 μg) subunits. Whole-cell patch-clamp recordings in HEK293T cells showed that fluorescently tagged GABA A receptors have similar dose-response curves of GABA as compared to untagged ion channels 17 . The coverslips were mounted using fluoromount-G (VWR, catalog #: 100502-406) and sealed.…”

“…Presumably, binding of Hispidulin and TP003 to the α1(G251D) variant improves stability and thus enhances the assembly and forward trafficking of GABA A receptors. We used Förster resonance energy transfer (FRET) assay to quantify the subunitsubunit assembly in HEK293T cells 17 . CFP-tagged α1(G251D) and YFP-tagged β2 exhibited a FRET efficiency of 21.4 ± 1.4%; Hispidulin or TP003 treatment significantly increased the FRET efficiency to 42.5 ± 1.2% and 41.0 ± 2.1%, respectively (Fig.…”

“…The human GABA A receptor α1 subunit missense mutations (S76R, R214C, D219N, G251D, P260L, M263T, T289P, and A322D) were constructed using QuikChange II site-directed mutagenesis Kit (Agilent Genomics, catalog #: 200523). Enhanced cyan fluorescent protein (CFP) was inserted between Lys364 and Asn365 in the TM3-TM4 intracellular loop of the α1 subunit, and enhanced yellow fluorescent proteins (YFP) was inserted between Lys359 and Met360 in the TM3-TM4 intracellular loop of the β2 subunit by using the GenBuilder cloning kit (GenScript, catalog #: L00701), as previously described 17 . All cDNA sequences were confirmed by DNA sequencing.…”

“…Pixel-by-pixel based sensitized acceptor FRET microscopy was performed as described previously 17,64 . For FRET experiments on GABA A receptors, (1) for FRET pair samples, HEK293T cells on coverslips were transfected with α1-CFP (donor) (0.5 μg), β2-YFP (acceptor) (0.5 μg), and γ2 (0.5 μg) subunits; (2) for the donor-only samples, to determine the spectral bleed-through (SBT) parameter for the donor, HEK293T cells were transfected with α1-CFP (donor) (0.5 μg), β2 (0.5 μg), and γ2 (0.5 μg) subunits; (3) for the acceptor-only samples, to determine the SBT parameter for the acceptor, HEK293T cells were transfected with α1 (0.5 μg), β2-YFP (acceptor) (0.5 μg), and γ2 (0.5 μg) subunits.…”

“…Then BiP (binding immunoglobulin protein) and calnexin in the ER act as profolding chaperones to promote the productive folding of GABA A receptors 15,16 . Furthermore, Hsp47 (heat shock protein 47) acts after BiP to interact with relatively folded subunits to enhance the subunit assembly process into pentameric receptors in the ER 17 . Then the receptors engage the trafficking receptors, such as LMAN1 (lectin, mannose binding 1) 18 , for transport to the Golgi and plasma membrane.…”