Site-Specific Incorporation of Genetically Encoded Photo-Crosslinkers Locates the Heteromeric Interface of a GPCR Complex in Living Cells

Abstract

Highlights d Photoactivatable unnatural amino acids inform the structural interface of 5-HT 2A R-mGluR2 d TAG mGluR2 constructs were co-expressed with 5-HT 2A R for photo-crosslinking d UV-induced crosslinking only in cells co-expressing 5-HT 2A R and mGluR2-TAG 4.44 d 5-HT 2A R interacts with mGluR2 via the intracellular end of mGluR2's TM4

Binding of Natural Peptide Ligands to the Neuropeptide Y5 Receptor

“…All R 35 Aa nalogues display ag reater than 100-fold loss of potency,w hich is the largest effect of all tested analogues and supports the importance of the positively charged residue for ligand recognition (Figure 8and Supporting Information, Table S4). Substitution of Y 36 to alanine yielded agreater than 40-fold increase in the EC 50 value for all the peptides.D ifferences among NPY,P YY and PP were found for position 26 and 27. Exchange of His 26 of NPY or PYY did not much affect the activity of the agonists (1-fold and 2-fold loss,respectively), whereas replacement of Arg 26 of PP induced a5 -fold activity loss.B yc ontrast, substitution of position 27 seriously influenced the potencyo fN PY (7-fold…”

“…[25,34] Most recently,the method has been expanded to study GPCR-arrestin interactions [35] and GPCR oligomerization. [36] Photo-crosslinking mapping of Y 5 Rw ith Azi yielded crosslinking hits in three well-defined regions of the receptor:T he N-terminus,ECL2, and ECL3.…”

“…Genetically encoded crosslinkers [30] have been widely applied to investigate peptide‐GPCR interactions [31] both at rhodopsin‐like [32, 33] and secretin‐like GPCRs [25, 34] . Most recently, the method has been expanded to study GPCR‐arrestin interactions [35] and GPCR oligomerization [36] . Photo‐crosslinking mapping of Y 5 R with Azi yielded crosslinking hits in three well‐defined regions of the receptor: The N‐terminus, ECL2, and ECL3.…”

“…Likewise,afree-acid derivative of NPY lacking just the amidation (NPY-COOH) showed a4 7-fold loss of potencya nd no additional reduction at the Q 3.32 Am utant (Figure 6B). We examined then the interactions of the side chain of Y 36 .Strikingly,while substitution of Y 36 with Ala lead to a4 3-fold potencyl oss at wt Y 5 R, substitution with amino acids of increasing size and hydrophobicity (series Ala, Leu, Phe,T rp) gradually rescued the ligand activity,with W 36 -NPY displaying wt-like potency( 2-fold loss) (Figure 3C). On the other hand, when receptor residues lying close to Y 36 (L 4.60 , T 5.39 ,W 6.48 ,F 7.31 ,Y 7.35 ,H 7.39 )w ere mutated to Ala, these receptor variants were activated (although in some cases with reduced potency) by NPY,b ut not by [Y 36 A]-NPY (Supplementary Table S2A highlighted lanes).…”

Spanning the gap: unraveling RSC dynamics in vivo

“…A growing number of recent reports have revealed the versatility of an in vivo approach to protein–protein interaction studies utilizing UV-activatable unnatural amino acid (UAA) crosslinkers (Coin et al 2013 ; Wagner 2020 ; Shah 2020 ). These UAAs are incorporated into proteins of living cells through the heterologous expression of an orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA CUA pair in the host organism (Fig.…”