Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane

doi:10.21203/rs.3.rs-1986998/v1

Download PDF

Article

Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane

https://doi.org/10.21203/rs.3.rs-1986998/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 13 Feb, 2023

Read the published version in Scientific Reports →

You are reading this latest preprint version

The mechanotransduction (MET) channel of the inner ear sensory receptor cells, termed hair cells, is a central protein complex that enables our senses of hearing and balance. Hair cell MET requires an elaborate interplay of multiple proteins that form the MET channel. One of the MET complex components is the transmembrane protein LHFPL5, which is required for hair cell MET and hearing. LHFPL5 is thought to interact with two other MET channel complex proteins, PCDH15 and TMC1. Despite localizing to the plasma membrane of stereocilia, the mechanosensing organelles of hair cells, LHFPL5 requires its binding partner within the MET complex, PCDH15, to localize to the stereocilia tips in hair cells and to the plasma membrane in heterologous cells. Using the Aquaporin 3-tGFP reporter (AGR) for plasma membrane localization, we found that a region within extracellular loop 1, which interacts with PCDH15, precludes the trafficking of the AGR reporter to the plasma membrane of HEK293 cells. Our results suggest that the presence of protein partners may mask endoplasmic reticulum retention regions or enable the proper folding and trafficking of the MET complex components, to facilitate expression of the MET complex at the stereocilia membrane.

In the inner ear, sensory hair cells use mechano-electrical transduction (MET) to convert mechanical forces into electrical signals, initiating the transmission of auditory and vestibular information. The correct targeting of MET pore forming transmembrane channel-like 1 (TMC1) protein to the tips of shorter stereocilia, the site of MET, as well as the proper function of MET channels rely on an alliance of multiple proteins that assemble the MET channel complex^1,2. One of the components of the MET channel complex is the Lipoma HMGIC (high mobility group protein I-C, now known as HMGA2) fusion partner-like (LHFPL5). LHFPL5, which was previously known as TMHS (tetraspan membrane protein of hair cell stereocilia) is necessary for proper MET channel function³. LHFPL5 is a four transmembrane segment protein from the superfamily of tetraspan proteins that includes the claudin tight junction proteins, gap junction proteins, peripheral myelin proteins, and ion channel auxiliary subunits⁴. Mutations in LHFPL5 may impair auditory and vestibular function in zebrafish, cause congenital hearing loss in hurry-scurry mice, and DFNB67 deafness in humans^5–8.

The peak of expression of LHFPL5 in the murine inner ear is between P0 and P3 corresponding with the period of stereocilia formation and MET acquisition^9,10. LHFPL5 is detected on the apical hair cell membrane and stereocilia of both inner and outer hair cells, and in hair cells of the vestibular maculae and cristae^9,11. Immunogold labeling revealed that LHFPL5 is present in hair bundles at P0 with the expression level peak at about P3¹⁰. LHFPL5 localizes to the tips of predominantly shorter stereocilia, the site of MET channels, and transiently to the kinocilium and lower shaft and ankle link areas¹⁰.

Initially, LHFPL5 was thought to be involved in the formation of hair cell stereocilia and hair bundle morphogenesis due to its spatial and temporal expression pattern. However, co-immunoprecipitation experiments and structural studies found that LHFPL5 interacts with another MET channel complex component protocadherin-15 (PCDH15)^3,12, pointing out to a role in MET for LHFPL5. Interestingly, LHFPL5 fails to localize to the plasma membrane of cultured cells and to the stereocilia tips in the absence PCDH15^3,10, suggesting that the formation of an LHFPL5-PCDH15 complex is necessary for proper LHFPL5 localization. PCDH15 contains a single transmembrane helix and its intracellular C-terminus interacts with the N-terminus of TMC1^12,13. Earlier reports demonstrated no evidence for co-immunoprecipitation of LHFPL5 by TMC1¹⁴, but a more recent study detected an interaction between the two proteins using co-immunoprecipitation experiments in heterologous expression systems¹⁵. It is possible that LHFPL5 may stabilize the interaction between PCDH15 and TMC1; in the absence of TMC1, PCDH15 and LHFPL5 are still targeted to stereocilia³, while pathogenic mutations of PCDH15 or LHFPL5 affect targeting of TMC1 to the stereocilia¹⁴.

LHFPL5, thus, resembles other ion channel regulatory subunits such as the TARPs of AMPA receptors that facilitate channel transport and regulate the properties of pore-forming channel subunits, but the LHFPL5 function remains unknown¹⁶.

LHFPL5 expressed in cultured cells consistently fails to localize to the plasma membrane and is retained intracelularly³. The failure of LHFPL5 to traffic to the plasma membrane in a heterologous system is a major obstacle to identifying and studying the function of this protein. Interestingly, co-expression with PCDH15 has been shown to allow for plasma membrane expression of both proteins, LHFPL5 and PCDH15³, suggesting that intracellular assembly of protein complexes may be indispensable for proper localization and function of these MET channel proteins.

Here we identified the LHFPL5 regions as responsible for its intracellular retention. We took advantage of the ability of Aquaporin 3-tGFP (AQP3-tGFP), which displays intense plasma membrane labeling in heterologous systems, to identify LHFPL5 sequences that prevent the plasma membrane localization of AQP3-tGFP. Using AQP3-tGFP based reporter (AGR), we identified two regions within LHFPL5 that preclude its expression at the plasma membrane of HEK293 cells: the extracellular loop 1 (ECL1) and transmembrane (TM) segment 4. Identifying the regions of LHFPL5 that preclude this protein from localizing to the plasma membrane will aid future studies aimed at reconstitution of the MET complex proteins at the plasma membrane in heterologous cells.

AGR reveals amino acid sequence LHFPL5 ^47–96 prevents trafficking to the plasma membrane in a heterologous cell line

The LHFPL5 gene encodes a membrane protein of approximately 24 kDa. The recent cryo-EM structure of the LHFPL5 in complex with PCDH15 (PDB ID: 6C14) revealed the LHFPL5 structure¹². LHFPL5 presents intracellular N- and C-terminal domains and four transmembrane segments resulting in two extracellular loops (ECL1 and ECL2) and one short intracellular loop (ICL)¹⁷. Employing the AQP3-tGFP Reporter (AGR) system^18,19, we examined all LHFPL5 extracellular and intracellular loops, as well as both termini, to identify regions that have the ability to preclude protein trafficking to the plasma membrane. As shown in Fig. 1, each region was individually tested using the AGR system. Fused to the C-terminus of AQP3-tGFP, specific LHFPL5 regions were transiently expressed in HEK293 cells. The N-terminal domain LHFPL5^1–23, ICL (LHFPL5^120–125), ECL2 (LHFPL5^149–176), and C-terminal domain LHFPL5^200–219 did not prevent trafficking to the PM when transiently expressed as a part of the AGR system in HEK293 cells (Fig. 1A, C-E, white arrows). On the contrary, the LHFPL5 fragment that encompasses amino acid residues 47–96 (LHFPL5^47–96), which reside between the first and second transmembrane segment, does not show any detectable plasma membrane (PM) localization (Fig. 1B), suggesting that the LHFPL5^47–96 region precludes expression of LHFPL5 at the PM.

E-Cadherin- and CD10-based specific reporters corroborate results obtained using AGR system

As a more specific alternative to the AGR system, we have designed a type I transmembrane protein-based E-Cadherin-mGFP (monomeric GFP) plasma membrane reporter (Fig. 2A). E-Cadherin contains a single transmembrane segment (TM) and intracellular C‐terminal domain, and it displays intense PM staining when transiently expressed as a fusion protein with mGFP in HEK293 cells. Therefore, the E-Cadherin‐mGFP reporter is a tool to examine endoplasmic reticulum retention regions in intracellular domains. CD10, a type II transmembrane protein, also displays intense plasma membrane staining when tagged with mGFP, but possesses the opposite topology of E‐Cadherin and AQP3: CD10 contains a single TM segment and extracellular C‐terminal domain (Fig. 2B), and thus, mGFP‐CD10 is used here to confirm whether extracellular domains contain any endoplasmic reticulum retention activity.

We used the E-Cadherin-mGFP and mGFP‐CD10 reporters to confirm that retention of LHPL5^47–96 was not due to the intracellular PM orientation imposed by the AGR system. The intracellular regions of LHFPL5 were fused to the C-terminus of E-Cahderin-mGFP, while the extracellular loops of LHFPL5 were attached to the C-terminus of mGFP-CD10 imitating their biological orientation in full length LHFPL5. As shown in Fig. 3, mGFP-CD10-LHPL5^47–96 displayed predominantly ER retention while E-Cadherin-mGFP-LHFPL5^1–23, E-Cadherin-mGFP- LHFPL5^120–125, mGFP-CD10-LHFPL5^149–176 and E-Cadherin-mGFP-LHFPL5^200–219 displayed PM localization, in agreement with our AGR results (Fig. 1).

Fragment sequences of LHFPL5^47–96 (ECL1) do not block trafficking to the PM

Next, we tested whether the ECL1 of LHFPL5 contains an Omega-type signal sequence that abrogates detectable PM localization^18,19. To narrow down the exact region of the potential signal, we split ECL1 of LHFPL5 encompassing amino acids LHFPL5^47–96 into four overlapping fragments. To examine whether any ECL1 fragments prevent trafficking to the PM, fragments were fused to the C-terminus of the AGR system and expressed in HEK293. As shown in Fig. 4, we detected PM localization of LHFPL5^47–62, LHFPL5^59–74, LHFPL5^71–86 or LHFPL5^83–96, suggesting that most of the LHFPL5^47–96 sequence is required for its ability to halt trafficking to the PM.

The Transmembrane Segments of LHFPL5 did not prevent trafficking to the PM

Since transmembrane segments have been shown to participate in ER retention in certain proteins²⁰, we used the AGR system to determine whether any of the four TM segments of LHFPL5 contribute to ER retention of LHFPL5 when expressed heterologously in HEK293 cells. Using the same systematic approach described above, we C-terminally attached each TM sequence to AGR. The resulting constructs, AQP3-tGFP-LHFPL5^16–45, AQP3-tGFP-LHFPL5^95–121, AQP3-tGFP-LHFPL5^126–154, or AQP3-tGFP-LHFPL5^180–201 were transiently transfected into HEK293 cells. As shown in Fig. 5, PM localization was not prevented by any of the four TM sequences. These results suggests that none of the TMs of LHFPL5 contain a strong ER retention signal that hinders PM localization in heterologous cells.

Hair cell MET channel complex component LHFPL5 is necessary for proper MET channel function. Mutations in LHFPL5 may impair hair cell MET, leading to auditory and vestibular deficits in zebrafish, causing hearing loss and vestibular dysfunction in hurry-scurry mice, and autosomal recessive nonsyndromic hearing loss in humans (DFN67)^5–8. LHFPL5 has been shown to interact with PCDH15, another MET channel complex component, and fails to localize to the tips of stereocilia in the absence of PCDH15^3,12. Moreover, LHFPL5 expressed in heterologous cells is retained intracellularly. The failure of LHFPL5 to traffic to the PM in a heterologous system is an obstacle to identifying and studying the function of this protein and its fundamental role in hearing. Here, using AGR and two additional novel reporters, E-Cadherin-mGFP and mGFP-CD10, we revealed the ability of the ECL1 region of LHFPL5 to halt trafficking to the PM in a heterologous cell line. When we examined the additional LHFPL5 regions using the AGR system (Fig. 1), including the remaining intracellular and extracellular loops, both N- and C-termini, as well as the four TM segments, none prevented LHFPL5 localization to the PM. These results were confirmed with a couple of novel reporters, which are based on type I transmembrane protein E-Cadherin or type II transmembrane protein CD10 (Figs. 2 and 3). These novel reporters are more specific to the protein region of interest since they will express the LHFPL5 regions in their intrinsic cellular orientation. Our data also expose the value of the AGR method and suggest that more specific E-Cadherin and CD10 based novel reporters may be sufficient for future experiments. We attempted to identify any shorter sequence within the ECL1 region of LHFPL5^47–96 responsible for preventing trafficking to the PM by examining four overlapping ECL1 fragments. None of the fragments retained the ability to prevent reporter trafficking to the PM; all AGR reporter containing fragments of ECL1 sequence reached the PM when expressed in HEK293 cells (Fig. 4), suggesting that most of the ECL1 region is necessary for halting trafficking to the PM. Furthermore, each TM segment of LHFPL5 was assessed for their potential role in intracellular retention. However, none of the AGR construct containing each TM segment trafficked to the PM (Fig. 5), suggesting a minor role for these protein regions in LHFPL5 intracellular retention. Thus, using the AGR system, we found that the ECL1 region LHFPL5^47–96 was the only LHFPL5 fragment that completely abrogated any detectable PM localization. Interestingly, the ECL1 contains the region known to interact with PCDH15¹². Thus, our results further suggest that the presence of cell-specific protein partners may mask ER-retention regions and facilitate trafficking to the PM in stereocilia¹⁹

Except for a small deletion resulting in a frameshift (p.31fs)²¹, deafness mutations in LHFPL5 do not localize to interaction sites of LHFPL5 and PCDH15¹², suggesting that these mutations do not alter the LHFPL5-PCDH15 interaction. As LHFPL5 Y127C, C161F, T165M and R176L deafness-causing mutants interact with PCDH15³, it is expected that LHFPL5 interacts with components of the mechanotransduction complex at different sites as well^3,12,14.

LHFPL5 protein structure resembles that of claudin proteins, sharing common features. The two cysteine residues in the extracellular ECL1 spaced 9–11 residues apart that are conserved in the claudin proteins are also present in LHFPL5. Additionally, LHFPL5 present the claudin signature sequence GLW within the ECL1. Mutations of these cysteine residues abolish ligand binding and the ability of claudins to form an ion barrier, suggesting an essential role in the correct folding of the ECL1^22,23. Furthermore, the tryptophan and arginine residues located at the beginning and end of the ECL1 are conserved in claudins and hypothesized to have an essential role in protein folding²⁴. This putative sequence is believed to be essential for the correct folding of the loop 1, ligand recognition, and trans interaction between two claudins expressed in various cell types^24–26.

Future studies will be aimed at finding the exact sequence of the omega-type signal within ECL1 and whether targeted mutagenesis of key residues can rescue LHFPL5 trafficking to the PM in heterologous cells.

Plasmid constructs. The AQP3-tGFP-pcDNA5/FRT plasmid, which is abbreviated here as AQP3-tGFP and termed as the AGR system, was synthesized as previously described^18,19. Lhfp5 based sequences based on Lhfp5 (NP_872354) were subcloned into the AQP3-GFP-pcDNA5/FRT plasmid using Hpa1 and EcoRV restriction sites, synthesized by Genscript (Piscatawas, NJ), and confirmed by sequencing.

Cell culture, Transfection. HEK293 cells were purchased from Life Sciences (Carlsbad, CA) and seeded in 12-well glass-bottom plates (Cellvis, CA), cultured in DMEM/F12 media (Life Technologies, CA) supplemented with 10% Fetal Bovine Serum and 1% pen/strep. Cells were transfected as previously described^18,19. Briefly, on the day of transfection, spent media was replaced by fresh media and HEK293 cells were transfected with plasmid constructs using Expi293fectamine (Life Technologies, CA). Red Fluorescent Protein based BacMam 2.0 constructs, specific for ER, were co-transfected the same day following manufacturer’s instructions (Thermo Fisher Scientific, MA). To label the cell nucleus, Hoechst staining was used (Life Technologies, CA). Experiments were performed 48h after transfection. Transfected HEK293 cells were examined with an SP8 confocal fluorescence microscope using 63× 1.4 NA objective, Leica (Wetzlar, Germany). We captured 3 to 6 fields of view per dish, using two dishes per construct in every experiment. All experiments were performed in duplicates on different days to ensure reproducibility.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Acknowledgements
We acknowledge use of the Leica SP8 microscope in the Light Microscopy Imaging Facility at Case Western Reserve University made available through the Office of Research Infrastructure (NIH) Shared Instrumentation Grant S10OD016164. This research was partly supported by the Center for Clinical Research and Technology at University Hospitals Cleveland Medical Center (R.S.) and partly by NIH grant DC015016 (R.S.).

Corey, D. P., Akyuz, N. & Holt, J. R. Function and Dysfunction of TMC Channels in Inner Ear Hair Cells. Cold Spring Harbor perspectives in medicine 9, doi:10.1101/cshperspect.a033506 (2019).
Cunningham, C. L. & Muller, U. Molecular Structure of the Hair Cell Mechanoelectrical Transduction Complex. Cold Spring Harbor perspectives in medicine 9, doi:10.1101/cshperspect.a033167 (2019).
Xiong, W. et al. TMHS is an integral component of the mechanotransduction machinery of cochlear hair cells. Cell 151, 1283-1295, doi:10.1016/j.cell.2012.10.041 (2012).
Charrin, S., Jouannet, S., Boucheix, C. & Rubinstein, E. Tetraspanins at a glance. J Cell Sci 127, 3641-3648, doi:10.1242/jcs.154906 (2014).
Longo-Guess, C. M. et al. A missense mutation in the previously undescribed gene Tmhs underlies deafness in hurry-scurry (hscy) mice. Proc Natl Acad Sci U S A 102, 7894-7899, doi:10.1073/pnas.0500760102 (2005).
Shabbir, M. I. et al. Mutations of human TMHS cause recessively inherited non-syndromic hearing loss. J Med Genet 43, 634-640, doi:10.1136/jmg.2005.039834 (2006).
Kalay, E. et al. Mutations in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene cause autosomal recessive nonsyndromic hearing loss. Hum Mutat 27, 633-639, doi:10.1002/humu.20368 (2006).
Erickson, T., Pacentine, I. V., Venuto, A., Clemens, R. & Nicolson, T. The lhfpl5 Ohnologs lhfpl5a and lhfpl5b Are Required for Mechanotransduction in Distinct Populations of Sensory Hair Cells in Zebrafish. Front Mol Neurosci 12, 320, doi:10.3389/fnmol.2019.00320 (2019).
Longo-Guess, C. M., Gagnon, L. H., Fritzsch, B. & Johnson, K. R. Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation. Mamm Genome 18, 646-656, doi:10.1007/s00335-007-9049-x (2007).
Mahendrasingam, S., Fettiplace, R., Alagramam, K. N., Cross, E. & Furness, D. N. Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15. PloS one 12, e0185285, doi:10.1371/journal.pone.0185285 (2017).
Li, X. et al. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice. FASEB J 33, 6838-6851, doi:10.1096/fj.201802155RR (2019).
Ge, J. et al. Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5. Elife 7, doi:10.7554/eLife.38770 (2018).
Maeda, R. et al. Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2. Proc Natl Acad Sci U S A 111, 12907-12912, doi:10.1073/pnas.1402152111 (2014).
Beurg, M., Xiong, W., Zhao, B., Muller, U. & Fettiplace, R. Subunit determination of the conductance of hair-cell mechanotransducer channels. Proc Natl Acad Sci U S A 112, 1589-1594, doi:10.1073/pnas.1420906112 (2015).
Yu, X. et al. Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding. Proc Natl Acad Sci U S A 117, 29894-29903, doi:10.1073/pnas.2011147117 (2020).
Ballesteros, A., Fenollar-Ferrer, C. & Swartz, K. J. Structural relationship between the putative hair cell mechanotransduction channel TMC1 and TMEM16 proteins. eLife 7, doi:10.7554/eLife.38433 (2018).
Suzuki, H. et al. Crystal structure of a claudin provides insight into the architecture of tight junctions. Science 344, 304-307, doi:10.1126/science.1248571 (2014).
Soler, D. C. et al. A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line. Sci Rep 11, 13673, doi:10.1038/s41598-021-93117-8 (2021).
Soler, D. C. et al. An uncharacterized region within the N-terminus of mouse TMC1 precludes trafficking to plasma membrane in a heterologous cell line. Sci Rep 9, 15263, doi:10.1038/s41598-019-51336-0 (2019).
Wang, J. M. et al. A transmembrane motif governs the surface trafficking of nicotinic acetylcholine receptors. Nat Neurosci 5, 963-970, doi:10.1038/nn918 (2002).
Bensaid, M. et al. DFNB66 and DFNB67 loci are non allelic and rarely contribute to autosomal recessive nonsyndromic hearing loss. Eur J Med Genet 54, e565-569, doi:10.1016/j.ejmg.2011.07.003 (2011).
Gunzel, D. & Yu, A. S. Claudins and the modulation of tight junction permeability. Physiol Rev 93, 525-569, doi:10.1152/physrev.00019.2012 (2013).
Colegio, O. R., Van Itallie, C., Rahner, C. & Anderson, J. M. Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture. Am J Physiol Cell Physiol 284, C1346-1354, doi:10.1152/ajpcell.00547.2002 (2003).
Krause, G. et al. Structure and function of extracellular claudin domains. Ann N Y Acad Sci 1165, 34-43, doi:10.1111/j.1749-6632.2009.04057.x (2009).
Evans, M. J. et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801-805, doi:10.1038/nature05654 (2007).
Mrsny, R. J. et al. A key claudin extracellular loop domain is critical for epithelial barrier integrity. Am J Pathol 172, 905-915, doi:10.2353/ajpath.2008.070698 (2008).

No competing interests reported.

Download PDF

Journal Publication

published 13 Feb, 2023

Read the published version in Scientific Reports →

Editorial decision: Major revision
20 Sep, 2022
Reviews received at journal
09 Sep, 2022
Reviewers agreed at journal
30 Aug, 2022
Reviewers invited by journal
30 Aug, 2022
Editor assigned by journal
30 Aug, 2022
Editor invited by journal
29 Aug, 2022
Submission checks completed at journal
29 Aug, 2022
First submitted to journal
22 Aug, 2022

You are reading this latest preprint version

Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

AGR reveals amino acid sequence LHFPL5 ^47–96 prevents trafficking to the plasma membrane in a heterologous cell line

E-Cadherin- and CD10-based specific reporters corroborate results obtained using AGR system

Fragment sequences of LHFPL5^47–96 (ECL1) do not block trafficking to the PM

The Transmembrane Segments of LHFPL5 did not prevent trafficking to the PM

Discussion

Materials And Methods

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

AGR reveals amino acid sequence LHFPL5 47–96 prevents trafficking to the plasma membrane in a heterologous cell line

E-Cadherin- and CD10-based specific reporters corroborate results obtained using AGR system

Fragment sequences of LHFPL547–96 (ECL1) do not block trafficking to the PM

The Transmembrane Segments of LHFPL5 did not prevent trafficking to the PM

Discussion

Materials And Methods

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

AGR reveals amino acid sequence LHFPL5 ^47–96 prevents trafficking to the plasma membrane in a heterologous cell line

Fragment sequences of LHFPL5^47–96 (ECL1) do not block trafficking to the PM