Technical Reference #1710

1710.

Calmodulin dynamically regulates the trafficking of the metabotropic glutamate receptor mGluR5 Jeong Ho Lee*; Jinu Lee†; Kyu Yeong Choi‡; Regine Hepp§; Jae-Youn Lee*; Mi Kyung Lim*; Mayumi Chatani-Hinze‡; Paul A. Roche§; Dong Goo Kim*; Young Soo Ahn*; Chul Hoon Kim*;¶; and Katherine W. Roche‡, Yonsei University College of Medicine, PNAS, 105(1710), (2008)
Link To Paper

Abstract:
Metabotropic glutamate receptors (mGluRs) 1–8 are G proteincoupled receptors (GPCRs) that modulate excitatory neurotransmission neurotransmitter release and synaptic plasticity. PKC regulates many aspects of mGluR function including protein–protein interactions Ca2 signaling and receptor desensitization. However the mechanisms by which PKC regulates mGluR function are poorly understood. We have now identified calmodulin (CaM) as a dynamic regulator of mGluR5 trafficking. We show that the major PKC phosphorylation site on the intracellular C terminus of mGluR5 is serine 901 (S901) and phosphorylation of this residue is up-regulated in response to both receptor and PKC activation. In addition S901 phosphorylation inhibits mGluR5 binding to CaM decreasing mGluR5 surface expression. Furthermore blocking PKC phosphorylation of mGluR5 on S901 dramatically affects mGluR5 signaling by prolonging Ca2 oscillations. Thus our data demonstrate that mGluR5 activation triggers phosphorylation of S901 thereby directly linking PKC phosphorylation CaM binding receptor trafficking and downstream signaling.

Keywords:
phosphorylation; protein kinase C; receptor trafficking

Materials & Methods:
TIRFM. HeLa cells transiently expressing wild-type mGluR5– mRFP or mGluR5–mRFP S901A were grown to 30% confluence on 40-mm glass bottom culture dishes (MatTek) washed in modified HBSS (140mMNaCl 10mMHEPES 11mMglucose 5 mM KCl 1 mM MgCl2 1 mM CaCl2 pH 7.4) and allowed to equilibrate for at least 30 min at 37°C before imaging. TIRFM was carried out by using a Nikon TIRF-2 microscope with TE2000-PFS and Chamber System (Nikon) and its temperaturecontrolled chamber was set at 37°C for live cell imaging. mGluR5–mRFP was excited with the 532-nm laser and illumination intensity was attenuated as needed by a neutral density filter. Incident illumination passing through the Apo TIRFM 100 1.49 NA oil-immersion objective (Nikon) was angled to obtain TIRFM images. The emitted fluorescence was captured by a C9100-12 Camera System (Hamamatsu Photonics). Before adding 500 M (final concentration) L-glutamate (Sigma) dissolved in modified HBSS the Perfect Focus System (PFS) unit (Nikon) was turned on to correct the focus drift caused by the addition of any reagents. MetaMorph 6.0 software (Universal Imaging Corp.) was used for image analysis. The data are presented as the percentages of integrated optical density of spots at each time point divided by the integrated optical density at time 0. Images were acquired every 3 s for 10 min with a 100-ms exposure time. mRFP or mGluR5–mRFP S901A were grown to 30% confluence on 40-mm glass bottom culture dishes (MatTek) washed in modified HBSS (140mMNaCl 10mMHEPES 11mMglucose 5 mM KCl 1 mM MgCl2 1 mM CaCl2 pH 7.4) and allowed to equilibrate for at least 30 min at 37°C before imaging. TIRFM was carried out by using a Nikon TIRF-2 microscope with TE2000-PFS and Chamber System (Nikon) and its temperaturecontrolled chamber was set at 37°C for live cell imaging. mGluR5–mRFP was excited with the 532-nm laser and illumination intensity was attenuated as needed by a neutral density filter. Incident illumination passing through the Apo TIRFM 100 1.49 NA oil-immersion objective (Nikon) was angled to obtain TIRFM images. The emitted fluorescence was captured by a C9100-12 Camera System (Hamamatsu Photonics). Before adding 500 M (final concentration) L-glutamate (Sigma) dissolved in modified HBSS the Perfect Focus System (PFS) unit (Nikon) was turned on to correct the focus drift caused by the addition of any reagents. MetaMorph 6.0 software (Universal Imaging Corp.) was used for image analysis. The data are presented as the percentages of integrated optical density of spots at each time point divided by the integrated optical density at time 0. Images were acquired every 3 s for 10 min with a 100-ms exposure time.

Microscopic Technique
Fluorescence Microscopy, Total Internal Reflection Fluorescence (TIRF)

Cell Type(s)
HeLa