EXPRESSION OF THE HUMAN 5-HYDROXYTRYPTAMINE(1A) RECEPTOR IN SF9 CELLS - RECONSTITUTION OF A COUPLED PHENOTYPE BY COEXPRESSION OF MAMMALIAN G-PROTEIN SUBUNITS

The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine(1A) (5-HT1A) receptor with mammalian G protein subunits was explored. The 5-HT1A receptor was found to assume an uncoupled phenotype when expressed alone in Sf9 cells at relatively high levels (5-34 pmol of receptor/mg of membrane protein), i.e. agonist-binding to the receptor was characterized by a relatively high K-d and an insensitivity to GTP, Go-expression of the receptor with members of the alpha(i) ''family'' together with various combinations of beta(1) and gamma subunits increased the affinity for agonists to that ob served for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5'-(beta,gamma-imino)triphosphate sensitivity. The agonists employed were [H-3]8-hydroxy-N,N-dipropyl-2-aminotetralin ([H-3] 8-OH-DPAT) and [I-125]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin ([I-125]8-OH-PI-PAT), The binding of an antagonist, [I-125]4-(2'-methoxyphenyl)-1-[2'-[N-(2''-pyridinyl)-p-iodobenzamido]ethyl] piperazine ([I-125]p-MPPI), was unaffected by co-expression of G protein subunits. Both alpha and beta gamma subunits were required for optimal coupling. No differences were evident among alpha(i1), alpha(i2), alpha(i3), alpha(o), and alpha(z) when expressed with beta(1) gamma(2) in this regard, nor among most permutations of beta(1) gamma subunits when expressed with alpha(i1), (beta(1) gamma(2) approximate to beta(1) gamma(3) approximate to beta(1) gamma(5) > beta(1) gamma(2)). alpha(s) and alpha(q) expressed with beta(1) gamma(2) did not participate in coupling, These data support the conclusion that normal interactions between a mammalian receptor and a select array of G proteins can be established in intact Sf9 cells, and extend previous observations of 5-HT1A receptor coupling to G(o) and the pertussis toxin-insensitive G protein G(z).