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Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane

Nature Neuroscience volume 9, pages 622–627 (2006)Cite this article

Abstract

Many neural signaling receptors are regulated by endocytosis, but little is known about receptor insertion into the plasma membrane. Time-lapse imaging of the β2 adrenergic receptor expressed in cultured rat hippocampal neurons, using pH-sensitive green fluorescent protein tagging and total internal reflection fluorescence microscopy, resolved distinct vesicular fusion events mediating receptor insertion into the somatodendritic plasma membrane. A 'transient' insertion mode resulted in rapid lateral dispersion of receptors immediately after insertion. A 'persistent' insertion mode resulted in the retention of inserted receptors in surface-accessible domains, which were relatively immobile for a prolonged 'wait' period before dispersing laterally. Distinct insertion modes were oppositely regulated by receptor activation and by mechanisms differing in their dependence on the signaling effector cyclic AMP–dependent protein kinase. These results reveal a new mechanism for homeostatic regulation of postsynaptic signaling and a 'kiss-and-wait' mode of regulated membrane protein insertion in neurons.

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Figure 1: SpH-β2AR insertion events in the cell body.
Figure 2: Time course and lateral dispersion of SpH insertion events from sites in the plasma membrane distinct from clathrin-coated pits.
Figure 3: SpH-β2AR insertion events in dendrites.
Figure 4: Regulation of SpH-β2AR surface insertion events by agonist.
Figure 5: Role of PKA in regulating transient SpH-β2AR insertion events.

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Acknowledgements

The authors thank R. Edwards, T. Ryan, N. Stuurman and R. Vale for valuable discussions; J. Rothman (Columbia University, New York) for providing pH-sensitive GFP constructs; W. Almers (Vollum Institute, Portland, Oregon) for the DsRed-tagged clathrin light chain construct; A. Douglass and A. Marley for constructing SpH-β2AR, and B. Lauffer (all University of California San Francisco, San Francisco) for β2AR antiserum; P. Herzmark for use of the objective warmer; and H. Bourne and R. Nicoll for critical comments on the manuscript. These studies were supported by research grants from the US National Institutes of Health and the National Institute on Drug Abuse. G.A.Y. was supported in part by the Pew Latin American Fellows Program.

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Authors and Affiliations

  1. Departments of Psychiatry and Cellular & Molecular Pharmacology, University of California San Francisco, 600 16th Street, San Francisco, 94143, California, USA

    Guillermo A Yudowski, Manojkumar A Puthenveedu & Mark von Zastrow

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Correspondence to Mark von Zastrow.

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Supplementary information

Supplementary Fig. 1

Functional integrity of SpH–B2AR and expression in cultured neurons. (PDF 51 kb)

Supplementary Fig. 2

Plasma membrane targeting, ligand–induced internalization and surface localization of SpH–B2AR in hippocampal neurons. (PDF 141 kb)

Supplementary Fig. 3

Recycling sequence–dependence and MES accessibility of SpH–B2AR insertion events. (PDF 75 kb)

Supplementary Methods (PDF 86 kb)

Supplementary Video 1

Sequential TIRF images from hippocampal neurons expressing SpH–B2AR and exposed to 10 μM isoproterenol for 10 minutes prior to imaging. Interval between frames is 100 ms. (QT 12880 kb)

Supplementary Video 2

A region of the plasma membrane showing examples of 'transient' (circled in red) and 'persistent' (circled in green) SpH–B2AR insertion events occurring in adjacent regions of the somatic plasma membrane. Hippocampal neurons were exposed to 10 μM isoproterenol for 10 minutes prior to sequential TIRF imaging (100 ms / frame). Trace below the movie shows peak pixel intensity in each circled region of the plasma membrane over time. (QT 13413 kb)

Supplementary Video 3

Example of a persistent SpH–B2AR insertion event occurring on a proximal dendrite. A number of transient insertion events are seen in the somatic plasma membrane also present in the image series. Hippocampal neurons were exposed to 10 μM isoproterenol for 10 minutes prior to sequential TIRF imaging (100 ms / frame). (QT 1403 kb)

Supplementary Video 4

Example of a transient SpH–B2AR insertion event occurring on a distal dendrite. Interval between frames is 100 ms. (QT 702 kb)

Supplementary Video 5

Transient SpH–B2AR insertion event in a distal dendrite (corresponding to that analyzed in Fig. 3 c and d). Interval between frames is 100 ms. (QT 242 kb)

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Yudowski, G., Puthenveedu, M. & von Zastrow, M. Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane. Nat Neurosci 9, 622–627 (2006). https://doi.org/10.1038/nn1679

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