Expression of the Human 5-Hydroxytryptamine1A Receptor in Sf9 Cells

Abstract

The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine1A (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 Kd and an insensitivity to GTP. Co-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 observed for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5'-(beta,gamma-imino)triphosphate sensitivity. The agonists employed were [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT) and [125I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl) amino]tetralin ([125I]8-OH-PIPAT). The binding of an antagonist, [125I]4-(2'-methoxyphenyl)-1-[2'-[N-(2"- pyridinyl)-p-iodobenzamido]ethyl]piperazine ([125I]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 approximately beta 1 gamma 3 approximately beta 1 gamma 5 > beta 1 gamma 1). 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 Gz.

The α2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different βγ Subunit Composition in Sf9 Insect Cell Membranes

“…Distinct, yet to be revealed, structural features within the G protein binding regions of receptor subtypes must also be taken into account in such a model. For example, a previous study showed that the 5HT 1A receptor interacts similarly with G proteins containing ␤ 1 ␥ 2 , ␤ 1 ␥ 3 , or ␤ 1 ␥ 5 dimers (8), whereas the present study revealed that the ␣ 2A -adrenergic receptor prefers G proteins containing the ␤ 1 ␥ 2 or ␤ 1 ␥ 3 dimer over that containing the ␤ 1 ␥ 5 dimer. Thus, the G protein binding pockets of the ␣ 2A -adrenergic receptor and the 5HT 1A may possess subtle structural differences that result in either more or less receptor to G␣␤␥ contact depending on the identity of the ␥ subunit.…”

“…To date, in vitro studies examining the contribution of a limited number of ␤␥ dimers to the specificity of receptor coupling have not yielded the same high degree of discrimination shown in the in vivo studies cited above (8,9). The present study extended this analysis to the ␣ 2A -adrenergic receptor and to ␤␥ dimers that represent the most extensive degree of structural diversity examined to date.…”

“…Statistical analysis revealed the ␤ 1 ␥ 1 , ␤ 1 ␥ 5 , and ␤ 1 ␥ 10 dimers supported the lowest levels of coupling. Previously, several groups have reported that the ␤ 1 ␥ 1 dimer was less effective than other ␤␥ dimers in supporting coupling to numerous receptors (8,9,30) and that this problem could not be overcome by increasing its concentration. To extend further these observations, we showed that increasing the concentration of the ␤ 1 ␥ 5 dimer did not raise the level of receptor coupling (Fig.…”

“…The present study extended this analysis to the ␣ 2A -adrenergic receptor and to ␤␥ dimers that represent the most extensive degree of structural diversity examined to date. Since baculovirus expression has been shown to be an effective means for producing functional G protein subunits (10 -13) as well as G protein-coupled receptors (8,9,14,15), this system was used to measure the level of interaction between the recombinant ␣ 2A -adrenergic receptor expressed in Sf9 cell membranes and reconstituted in the presence or absence of purified G i proteins of varying ␤ or ␥ subtype composition. Among the two ␤ subtypes or eight ␥ subtypes tested, 30-fold differences were observed in their relative abilities to support coupling of the same ␣ subunit to the recombinant ␣ 2A -adrenergic receptor.…”

Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades

“…We chose to investigate the relationship between NHE and ERK in CHO-K1 fibroblasts which were transfected with the human 5-HT "A receptor, a prototypical G i α -linked receptor [26,27]. The relatively selective nature of the coupling of this receptor to G proteins would reduce potential ambiguities that might result from a more complex pattern of G protein coupling.…”