Abstract

DNA encoding the human alpha 2-C-10-adrenergic receptor was transfected into Rat-1 fibroblasts by CaPO4 precipitation, and clones expressing the receptor were isolated and expanded. One clone (1C) expressing high levels of the receptor was studied in order to determine the contacts between this receptor and guanine nucleotide-binding proteins (G proteins) mediating second messenger signaling. The alpha 2-adrenergic agonist UK 14304 stimulated high affinity GTPase activity in membranes from these cells. Incubation of these membranes with Protein A-purified fractions from an antiserum able to identify the carboxyl-terminal decapeptide common to Gi1 alpha and Gi2 alpha was partially able to prevent agonist stimulation of high affinity GTPase activity. Similar results were produced with an antiserum that identifies the carboxyl-terminal decapeptide of Gi3 alpha. In contrast, equivalent fractions of antisera that identify the carboxyl-terminal decapeptides of Go alpha and Gs alpha did not inhibit receptor stimulation of high affinity GTPase activity. Coincubation of the membranes from the cells with Protein A-purified fractions from the anti-Gi1 alpha + Gi2 alpha antiserum and the anti-Gi3 alpha antiserum produced greater inhibition of UK14304-stimulated GTPase activity than did either of the two antisera in isolation. These data show direct interaction of the human alpha 2-C10-adrenergic receptor, when expressed in this clone of Rat-1 fibroblasts, with multiple pertussis toxin-sensitive G proteins and demonstrate that a single receptor has the physical capacity to interact functionally with more than a single pertussis toxin-sensitive G protein in a native membrane. Furthermore, because the two antisera were able to inhibit receptor stimulation of high affinity GTPase activity to similar degrees, the G protein pools identified by these antisera must contribute similar amounts of the total receptor activation of pertussis toxin-sensitive G proteins in these cells.