Functional Domains of the Mouse β₃-Adrenoceptor Associated with Differential G Protein Coupling

Abstract

Alternative splicing of mouse ␤ 3 -adrenoceptor transcripts produces an additional receptor isoform (␤ 3b -adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (␤ 3a -adrenoceptor). We have shown that the ␤ 3b -adrenoceptor couples to both G s and G i , whereas the ␤ 3a -adrenoceptor couples only to G s . To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant ␤ 3 -adrenoceptors. In Chinese hamster ovary cells expressing ␤ 3 -adrenoceptors truncated at the splicing point, cAMP accumulation withincreased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the ␤ 3a -adrenoceptor inhibits coupling to G i . We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing ␤ 3a -adrenoceptor, ␤ 3b -adrenoceptor, and the truncated ␤ 3 -adrenoceptor. Treatment with ␤ 3a -Tp10 (1 M) caused cAMP responses to CL316243 in the ␤ 3a -adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala ␤ 3a -adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant ␤ 3b -adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the ␤ 3a -adrenoceptor cannot couple to G i because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the ␤ 3a -adrenoceptor.

Interaction with Caveolin-1 Modulates G Protein Coupling of Mouse β3-Adrenoceptor

“…Expression of the Mouse ␤ 3a -and ␤ 3b -AR and Receptor Mutants in CHO-K1 Cells-Plasmids (pcDNA3.1ϩ) carrying the coding region for each of the ␤ 3a -and ␤ 3b -AR, a truncated ␤ 3 -AR, and the mutant Y392A were as described previously (21,22). Five additional mutants were created to examine the potential importance of residues in the C terminus for G protein coupling.…”

“…The ␤ 3a -and ␤ 3b -AR isoforms differ only in their distal C-terminal tail, yet cAMP accumulation mediated by the ␤ 3b -AR is increased following pretreatment of cells with pertussis toxin (PTX), whereas the ␤ 3a -AR response is PTX-insensitive. Use of cellpermeable peptides corresponding to the unique ␤ 3a -and ␤ 3b -AR C termini demonstrated that the ␤ 3a -AR C-terminal tail interacts with a distinct protein or signaling complex (22).…”

Relaxin Family Peptide Receptor (RXFP1) Coupling to Gαi3Involves the C-Terminal Arg752and Localization within Membrane Raft Microdomains

“…Mutation of Ser 755 partially changed the signaling profile of RXFP1, perhaps suggesting that it forms part of a binding motif involving Arg 752 . To examine RXFP1-G␣ i3 signaling further, we disrupted membrane rafts using filipin III (Sato et al, 2007) and showed that RXFP1 coupling to G␣ i3 was dependent upon their presence, whereas RXFP2 signaling was insensitive to membrane raft disruption. Indeed, RXFP2-10ct1 coupling to the G␣ i3 pathway was also prevented by membrane raft disruption, whereas deletion of the final 10 amino acids of the RXFP1 C terminus or substitution of Arg 752 for Ala removed the sensitivity of RXFP1 to membrane raft disruption.…”

“…We have previously shown that ␤ 3 -adrenoceptor signaling is influenced by the localization of the receptor within caveolae (Sato et al, 2007). Thus, we tested the effect of membrane raft disruption upon RXFP1 signaling using filipin III (Fig.…”

Ligand-Directed Signaling at the β3-Adrenoceptor Produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) Relative to Receptor Agonists

“…Likewise, ␤ 3 -AR-mediated Erk1/2 phosphorylation is independent of G i , because the response is insensitive to pertussis toxin, and ␤ 3a -AR do not couple to G i (Hutchinson et al, 2002;Sato et al, 2005).…”