Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway

Kenneth L Madsen,Thor S Thorsen,Troels Rahbek-Clemmensen,Jacob Eriksen,Ulrik Gether

doi:10.1074/jbc.m111.294702

Kenneth L Madsen, Thor S Thorsen + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111.294702

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Abstract

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

Highlights

The role of Protein interacting with C kinase 1 (PICK1) in regulating trafficking of its PSD-95/Discs large/ZO-1 (PDZ) domain binding partners (e.g. amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) receptors) remains unclear
When the last 24 residues from the C terminus of the dopamine transporter (DAT) were fused to the Tac C terminus (TacDAT C24) (Fig. 1a), we observed co-clustering with PICK1 in COS7 cells [44]
In one of our earlier studies, we did not see any convincing signs of clustering when DAT tagged at the N terminus with green fluorescent protein (GFP) was co-expressed with PICK1 [46]

Summary

Background

The role of PICK1 in regulating trafficking of its PDZ domain binding partners (e.g. AMPA receptors) remains unclear. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway. The data suggest that this role depends on the inherent postendocytic sorting properties of the binding partners; i.e. PICK1 clustered and inhibited recycling of binding partners that independently sorted to the Rab11-mediated “slow” or “long loop” recycling pathway. This further implies that PICK1 by itself neither resides in nor targets its PDZ cargo into a recycling pathway. The results support the notion of PICK1 as a highly versatile scaffolding protein capable of mediating cargo-determined distinct cellular functions depending on the nature and inherent properties of the PDZ domain binding partner

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION