Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells. Am J Physiol 1997 Jul;273(1 Pt 2):F129-35
Date
07/01/1997Pubmed ID
9249600DOI
10.1152/ajprenal.1997.273.1.F129Scopus ID
2-s2.0-0030774914 (requires institutional sign-in at Scopus site) 51 CitationsAbstract
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.
Author List
Arthur JM, Collinsworth GP, Gettys TW, Quarles LD, Raymond JRAuthor
John R. Raymond MD President, CEO, Professor in the President department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Affinity LabelsAmino Acid Sequence
Animals
Azides
Base Sequence
Calcium
Cattle
Cell Line
Cloning, Molecular
Cross-Linking Reagents
DNA Primers
Dogs
GTP-Binding Proteins
Gadolinium
Guanosine Triphosphate
Humans
Kidney
Molecular Sequence Data
Polymerase Chain Reaction
RNA, Messenger
Receptors, Calcium-Sensing
Receptors, Cell Surface
Transcription, Genetic