Caveolin-1 alters Ca(2+) signal duration through specific interaction with the G alpha q family of G proteins.
Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca(2+) signaling through the Galpha(q)-phospholipase-Cbeta (PLCbeta) pathway using Fischer rat thyroid cells that lack Cav1 (FRTcav(-)) and a sister line that forms caveolae-like domains due to stable transfection with Cav1 (FRTcav(+)). In the resting state, we found that eCFP-Gbetagamma and Galpha(q)-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Galpha(q)-eYFP and eCFP-Gbetagamma remains high in FRTcav(-) cells, but decreases almost completely in FRTcav(+) cells, suggesting that Cav1 is increasing the separation between Galpha(q)-Gbetagamma subunits. In FRTcav(-) cells overexpressing PLCbeta, a rapid recovery of Ca(2+) is observed after stimulation. However, FRTcav(+) cells show a sustained level of elevated Ca(2+). FRET and colocalization show specific interactions between Galpha(q) and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Galpha(q)-eGFP is unaffected by activation in either cell type. The mobility of eGFP-Gbetagamma remains slow in FRTcav(-) cells but increases in FRTcav(+) cells. Together, our data suggest that, upon stimulation, Galpha(q)(GTP) switches from having strong interactions with Gbetagamma to Cav1, thereby releasing Gbetagamma. This prolongs the recombination time for the heterotrimer, thus causing a sustained Ca(2+) signal.
Sengupta
Parijat
P
Department of Physiology and Biophysics, BST6-145, Stony Brook University, Stony Brook, NY 11794-8661, USA.
Philip
Finly
F
Scarlata
Suzanne
S
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D051242:Caveolin 1 / Q000378:metabolism*; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D051242:Caveolin 1; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D021122:Protein Subunits; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002217:Carbachol; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium