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Identification of clustered phosphorylation sites in PKD2L1: how PKD2L1 channel activation is regulated by cyclic adenosine monophosphate signaling pathway

  • Ion channels, receptors and transporters
  • Published:
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Abstract

Polycystic kidney disease 2-like-1 (PKD2L1), or polycystin-L or TRPP2, formerly TRPP3, is a transient receptor potential (TRP) superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. PKD2L1 has been reported to take part in hedgehog signaling in renal primary cilia and sour tasting coupling with PKD1L3. In addition to the previous reports, PKD2L1 is recently found to play a crucial role in localization with β2-adrenergic receptor (β2AR) on the neuronal primary cilia. The disruption of PKD2L1 leads to the loss of β2AR on the primary cilia and reduction in intracellular concentration of cyclic adenosine monophosphate (cAMP). Since the role of cAMP and PKA is frequently mentioned in the studies of PKD diseases, we investigated on the mechanism of cAMP regulation in relation to the function of PKD2L1 channel. In this study, we observed the activity of PKD2L1 channel increased by the downstream cascades of β2AR and found the clustered phosphorylation sites, Ser-682, Ser-685, and Ser-686 that are significant in the channel regulation by phosphorylation.

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Abbreviations

AC:

adenylate cyclase

ADPKD:

autosomal dominant polycystic kidney disease

AR:

adrenergic receptor

cAMP:

cyclic adenosine monophosphate

CMZ:

calmidazolium

FSK:

forskolin

ISO:

isoproterenol

PKA:

protein kinase A

PKD2L1:

polycystic kidney disease 2-like-1

TRP:

transient receptor potential

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Acknowledgments

We thank Dr. Markus Delling (UCSF) for kindly donating human PKD2L1 construct. This research project was supported by the BK21-plus education program of the MOE (Ministry of Education), Korea by the National Research Foundation of Korea, and Mid-career Researcher Program through NRF grant funded by the Korea government (Minstry of Science and ICT) (2015R1A2A1A05001756).

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  1. Department of Physiology, Seoul National University, College of Medicine, Biomedical Science Building 117, 103 Daehakro, Jongro-gu, Seoul, 110-799, South Korea

    Eunice Yon June Park, Misun Kwak & Insuk So

  2. Department of Physiology, University of California, San Francisco, CA, 94158-2517, USA

    Kotdaji Ha

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ESM 1

Supplementary Fig. 1 The potentiation and inactivation of the channel by different concentrations of the intracellular free calcium. a A full current trace of PKD2L1 measured under 16 nM free calcium condition in a PKD2L1 expressed HEK293 cell using whole-cell patch clamp technique. From the holding potential at − 60 mV, ramp pulse was applied from − 100 to 100 mV. b A full current trace of PKD2L1 measured under 100 nM free calcium condition in a PKD2L1 expressed HEK293. c The I-V relationship of PKD2L1. Each colored line indicates I-V relationship at the basal current amplitude (black), the peak amplitude in 16 nM free calcium condition (blue), and the peak amplitude in 100 nM free calcium condition (purple). Supplementary Fig. 2 Endogenous channel current in HEK293 cell. a A full current trace of HEK293 cell with b 1 μM CMZ, and c 100 μM ISO application. d A full current trace of HEK293 transfected with Gαs (Q227L). e A full current trace of HEK293 cell with 100 μM FSK and f 100 μM cAMP application. Supplementary Fig. 3 A summarized basal current amplitude of PKD2L1WT and PKD2L1 mutants. a A summarized basal current amplitude of PKD2L1 single mutants. b A summarized basal current amplitude of PKD2L1 double mutants. c A summarized basal current amplitude of PKD2L1 clustered mutants. *p < 0.05, **p < 0.01, ***p < 0.001. Supplementary Fig. 4 Double mutants activated by 1 μM CMZ and 100 nM free calcium solution a A full current trace of PKD2L1(S581A/S747A), b PKD2L1(S747A/S805A), and c PKD2L1(S581A/S805A) activated by 1 μM CMZ. d A summarized increased current amplitude of double mutants induced by 1 μM CMZ. e A full current trace of PKD2L1(S581A/S747A), f PKD2L1(S747A/S805A), and g PKD2L1(S581A/S805A) activated by 100 nM free calcium concentration. h A summarized peak current amplitude of double mutants induced by 100 nM free calcium concentration. Supplementary Fig. 5 The consistent surface expression level of PKD2L1WT and the double mutants. a Representative surface expression level of PKD2L1 and three double mutants. (PDF 1242 kb)

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Park, E.Y.J., Kwak, M., Ha, K. et al. Identification of clustered phosphorylation sites in PKD2L1: how PKD2L1 channel activation is regulated by cyclic adenosine monophosphate signaling pathway. Pflugers Arch - Eur J Physiol 470, 505–516 (2018). https://doi.org/10.1007/s00424-017-2095-7

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