Specific activation of G_s by synthetic peptides corresponding to an intracellular loop of the β‐adrenergic receptor

Abstract

Peptide= correspondinR, to the amino tLcid ~equenee or the hamster/~=.~drenergi¢ receptor If/eAR.) were synthesized and their ability to activate purified O.proteins determined. Two peptides. ¢omprismll the N, and C-terminal I$ m'nino arid= of the putative third intraeellular loop region or the p~AR were found to aetivale the G.protein O, but not to activate a preparation of G,/G,. Other p~ptides ¢orre~pondin= to the intern=l portion= of this loop and the C.terminal lab region fitiled to activate either G.protein. The presence of pho=pholipid vesicles was required for thil activation. The ob~:rvation that peptides with ~quences eorrespondinii to the ends of the third intraeelhflar loop of the,BAR can spccifcally activate O, co)~firms the results of prev=ous muta$enesis studies on tho receptor and demonstrates that the ~econdary strugttlre ¢onferred by the amino acid =equ=nces in these regions is sufficient for the activation of Q,proteins.

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

“…In addition, it is possible that the sequence proximal to the QKIDKSEGF motif also contributes to G s activation. Indeed, previous studies demonstrated that peptides from the proximal portion of the hamster ␤ 2 AR ICL3 are direct activators of nucleotide exchange on G s (52). The most effective peptide from these studies was VYS-RVFQVAKRQLQK, which is proximal to the QKIDKSEGF motif.…”

“…Although previous studies used unmodified peptides that have a limited ability to cross the cell membrane (52), the N-terminal palmitoylation and C-terminal amidation of a pepducin enable membrane incorporation and effective delivery to the intracellular surface of the cell membrane (18). For ICL3-8, this provides a means of targeting it to the site of action as G s is localized to the intracellular surface of the plasma membrane.…”

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

“…From the assignment of Lys-290 to the helical part this residue can be considered either as an integral part of the membrane or as part of a cytoplasmic extension of the transmembrane helix [2,27].…”

Agonist-induced conformational changes in the G-protein-coupling domain of the β 2 adrenergic receptor

“…Mutagenesis studies have demonstrated that sequences immediately carboxyl-terminal to Cys-265 are important for G-protein coupling (6 -10), and mutation of the nearby Leu-272 leads to constitutive activation (11). In addition, synthetic peptides representing sequences from the carboxyl-terminal portion of IC3 are capable of directly stimulating G proteins (12)(13)(14). Therefore, a f luorophore covalently bound to Cys-265 is well positioned to detect agonistinduced conformational changes relevant to G-protein activation.…”