Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis
Rita C. Andersen, … , Ulrik Gether, Kenneth L. Madsen
Rita C. Andersen, … , Ulrik Gether, Kenneth L. Madsen
Published January 25, 2022
Citation Information: J Clin Invest. 2022;132(5):e144904. https://doi.org/10.1172/JCI144904.
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Research Article Cell biology Metabolism

Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis

Abstract

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

Authors

Rita C. Andersen, Jan H. Schmidt, Joscha Rombach, Matthew D. Lycas, Nikolaj R. Christensen, Viktor K. Lund, Donnie S. Stapleton, Signe S. Pedersen, Mathias A. Olsen, Mikkel Stoklund, Gith Noes-Holt, Tommas T.E. Nielsen, Mark P. Keller, Anna M. Jansen, Rasmus Herlo, Massimo Pietropaolo, Jens B. Simonsen, Ole Kjærulff, Birgitte Holst, Alan D. Attie, Ulrik Gether, Kenneth L. Madsen

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Figure 5

Expression of PICK1-R158Q and PICK1-R247H in INS-1E cells reduces insulin storage.

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Expression of PICK1-R158Q and PICK1-R247H in INS-1E cells reduces insuli...
INS-1E cells were transiently transfected with YFP alone (ctrl) or YFP fused to PICK1 WT, R158Q, R185Q, R197Q, and R247H. (A) Representative confocal images of INS-1E cells immunostained for YFP (yellow), PICK1 (cyan), and insulin (magenta). Merged images show PICK1 and insulin immunosignals. Examples of transfected cells (YFP-positive) are outlined with yellow dotted lines, and untransfected cells are outlined with white dotted lines. Scale bar: 10 μm. (B) Quantified insulin and PICK1 immunosignals per cell correlate and can be fitted with a linear regression. The gray dotted line represents the linear regression for PICK1 WT. Expression of PICK1, R158Q, or R247H reduces the slope of the correlation line. (C) Quantification of the PICK1 immunosignal. R158Q (n = 159), R185Q (n = 119), R197Q (n = 125), R247H (n = 140), ctrl (n = 227) compared with WT (n = 188), with Kruskal-Wallis test followed by Dunn’s multiple-comparison test. Data are shown as mean ± SEM. (D) Quantification of the insulin immunosignal. *P < 0.05, **P < 0.01, ***P < 0.001, PICK1 coding variants compared with WT; ###P < 0.001, variants compared with ctrl; Kruskal-Wallis test followed by Dunn’s multiple-comparison test.