Human substance P receptor expressed in Sf9 cells couples with multiple endogenous G proteins.
To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [125I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [35S]GTPgammaS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [alpha-32P]-azidoanilido GTP ([alpha-32P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Galpha-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Galpha(o), Galpha(q/11), and Galpha(s). While hSPR's ability to associate with Gq/11 is well-documented, the present study provides the first evidence of hSPR's potential to activate Galpha(o) and Galpha(s).
Nishimura
K
K
Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA.
Frederick
J
J
Kwatra
M M
MM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D018040:Receptors, Neurokinin-1 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics* / Q000821:virology
D008565:Membrane Proteins; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins