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A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels.

ABSTRACT: Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ?50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 ? subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway.

SUBMITTER: Li X

PROVIDER: S-EPMC4946915 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels.

Li Xiangming X Ortega Bernardo B Kim Boyoung B Welling Paul A PA

The Journal of biological chemistry 20160523 29

Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that ...[more]

PMID: 27226616

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Project description:BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with beta2-adrenergic antagonist ICI 118,551 (100 microM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the beta-adrenergic agonist isoproterenol (10 microM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 microM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and beta-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results.

Dataset Information

A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels.

Publications

A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels.

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