Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells.
Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.
Arthur
J M
JM
Department of Medicine, University of Louisville, Kentucky 40292, USA.
Collinsworth
G P
GP
Gettys
T W
TW
Quarles
L D
LD
Raymond
J R
JR
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017931:DNA Primers; D004285:Dogs; D019204:GTP-Binding Proteins / Q000502:physiology*; D005682:Gadolinium / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000502:physiology*; D014158:Transcription, Genetic
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D017931:DNA Primers; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D005682:Gadolinium; D019204:GTP-Binding Proteins; D002118:Calcium