Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells
Ifie, E; Russell, M; Leete, P; et al.Dhayal, S; Morgan, N; Sebastiani, G; Nigi, L; Dotta, F; Marjomäki, V; Eizirik, D; Morgan, NG; Richardson, SJ
Date: 3 August 2018
Journal
Diabetologia
Publisher
Springer Verlag
Publisher DOI
Abstract
Aims/hypothesis The Coxsackie and adenovirus receptor (CAR) is a transmembrane celladhesion
protein that serves as an entry receptor for enteroviruses and may be essential for their
ability to infect cells. Since enteroviral infection of beta-cells has been implicated as a factor
that could contribute to the development of type 1 ...
Aims/hypothesis The Coxsackie and adenovirus receptor (CAR) is a transmembrane celladhesion
protein that serves as an entry receptor for enteroviruses and may be essential for their
ability to infect cells. Since enteroviral infection of beta-cells has been implicated as a factor
that could contribute to the development of type 1 diabetes, it is often assumed that CAR is
displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and
it is not known whether all isoforms subserve similar physiological functions. In the present
study, we have determined the profile of CAR isoforms present in human beta cells and
monitored the subcellular localisation of the principal isoform within the cells.
Methods Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals
and those with type 1 diabetes were studied. Immunohistochemistry, confocal
immunofluorescence, electron microscopy and western blotting with isoform-specific antisera
were employed to examine the expression and cellular localisation of the five known CAR
isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on
RNA extracted from isolated human islets.
Results An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was
expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV,
was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in
isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal
isoform in islets and was localised mainly within the cytoplasm of beta-cells, rather than at the
plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling,
consistent with its retention within a specific membrane-bound compartment. Coimmunofluorescence
analysis revealed significant co-localisation of CAR-SIV with zinc
transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not
3
proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin
secretory granules. Immunogold labelling and electron microscopic analysis confirmed that
CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no
immunolabelling of the protein was detected on the secretory granules of adjacent exocrine
cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with Ckinase
1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation
and trafficking.
Conclusions/interpretation: The SIV isoform of CAR is abundant in human beta cells and is
localised mainly to insulin secretory granules, implying that it may be involved in granule
trafficking and maturation. We propose that this subcellular localisation of CAR-SIV
contributes to the unique sensitivity of human beta cells to enteroviral infection.
Institute of Biomedical & Clinical Science
Collections of Former Colleges
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