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First published online 8 April 2008
doi: 10.1242/jcs.020081

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Caveolin-1 alters Ca²⁺ signal duration through specific interaction with the G_q family of G proteins

Parijat Sengupta, Finly Philip^* and Suzanne Scarlata

Department of Physiology and Biophysics, BST6-145, Stony Brook University, Stony Brook, NY 11794-8661, USA

View larger version (58K): [in this window] [in a new window]   Fig. 1. Distribution of Cav1-eGFP in FRT cells in the basal and stimulated states. (A) Confocal image of an FRT cell expressing Cav1-eGFP, showing typical punctuate structures. (B) Confocal image of FRT cells expressing a membrane marker, MEM-eYFP, showing uniform distribution. (C) z stack of confocal images (from bottom to top) illustrate the cellular distribution of Cav1-eGFP in different planes of FRT cells. Cav1-eGFP forms large punctuate structures (indicated by white arrows) on the membrane. The thickness of each slice is 1 µm. Scale bar: 5 µm. (D) A time series of fluorescence images showing that stimulation of FRT cells expressing Cav1-eGFP does not cause internalization of Cav1-eGFP when muscarinic receptors are stimulated using 5 µM carbachol. Scale bar: 2 µm. Total duration of time series is 330 seconds.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Gβ and Gq subunits dissociate upon carbachol stimulation in the presence of Cav1. (A) Pseudo-colored FRET images illustrating the time-dependence of energy transfer from eCFP-Gβ to Gq-eYFP in acetylcholine (5 µM)-stimulated FRTcav+ (upper panel) and FRTcav– (lower panel) cells. The FRET color-scale is shown for comparison (red, 100%; black, 0%). For both series, the first panel was recorded before stimulation and the elapsed time between each image is 20 seconds. (B) Compiled results of the changes in NFRET from eCFP-Gβ to Gq-eYFP in unstimulated and acetylcholine (5 µM)-stimulated FRTcav+ and FRTcav– cells with time. Studies were done using nine (FRTcav–) and four (FRTcav+) independent sets of samples (each measured in triplicate and averaged). (C) Pseudo-colored NFRET image illustrating energy transfer between free eCFP and eYFP.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Mobility of Gq and Gβ in FRTcav– and FRTcav+ cells. Box-and-whisker diagram of the diffusion coefficient of Gq-eGFP (left) and eGFP-Gβ (right) in FRTcav+ and FRTcav– cells in the basal and stimulated (5 µM carbachol) state measured by FCS. The data set from each experiment is represented as a separate box. The boundary of the box is determined by the 25th and 75th percentiles, and the whiskers are determined by the 5th and 95th percentiles. The small red square within each box indicates the value of the mean for each set of data (n=20-50 per set; every D is calculated using a set of six to ten measurements of 10 seconds each). Data was collected with a band-pass emission filter (BP505-550) with excitation at 488 nm. For stimulated cells, data was recorded for 8 minutes post-stimulation from different cells and then averaged. All measurements were performed at 25°C.

View larger version (28K): [in this window] [in a new window]   Fig. 4. The presence of Cav1 causes a sustained Ca2+ response. Change in intracellular Ca2+ in 1-ml suspensions of 106 FRTcav+ or FRTcav– cells transfected with either empty vector, PLCβ1 or PLCβ2, and loaded with the Ca2+-sensitive dye FURA-2AM upon addition of 1 µM acetylcholine (see Materials and Methods). n=6 of triplicate sets of samples. Black arrows indicate time of stimulus addition.

View larger version (76K): [in this window] [in a new window]   Fig. 5. Immunofluorescence confocal images showing the colocalization of Cav1 and Gq. Confocal images of fixed FRT cells expressing transiently transfected Cav1-eGFP (green) are immunostained for endogenous Gq (red). Yellow pixels in the merged images indicate colocalization. Cells were either fixed without stimulation (top) or at 4 minutes after stimulation with 5 µm carbachol (bottom). Images were recorded with emission filter sets BP505-530 (green) and LP650 (red) using two separate lasers for sequential excitation (488 nm for green and 633 nm for red).

View larger version (20K): [in this window] [in a new window]   Fig. 6. FRET illustrates that activated Gq has a stronger affinity for Cav1-eGFP than do Gβ subunits. (A) The titration curve of XRITC-Gβ and XRITC-Gq(GTPS), showing the normalized decrease in Cav1-eGFP intensity as the G proteins are added, caused by energy transfer (FRET). (B) Bar graph showing the values of the normalized donor intensities for free acceptor XRITC at 60 nM (n=2), XRITC-Gβ at 70 nM (n=3), XRITC-Gq(GDP) (n=3), XRITC-Gq(GTPS) (n=5), XRITC-Gq(GTPS) + 80 nM PLCβ1 (n=2) and XRITC-Gq (GTPS) starting with threefold the initial amount of Cav1-eGFP-containing FRTcav– membranes (n=3). Asterisks indicate significant difference, P<0.001, by one-way variance from control.

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