Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

Abstract

Dedicated to Horst Kunz on the occasion of his 80th birthday.Fluorescence microscopy imaging enables receptor proteins to be investigated within their biological context. A key challenge is to site-specifically incorporate reporter moieties into proteins without interfering with biological functions or cellular networks. Small peptide tags offer the opportunity to combine inducible labeling with small tag sizes that avoid receptor perturbation. Herein, we review the current state of live-cell labeling of peptide-tagged cell-surface proteins. Considering their importance as targets in medicinal chemistry, we focus on membrane receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We discuss peptide tags that i) are subject to enzyme-mediated modification reactions, ii) guide the complementation of reporter proteins, iii) form coiled-coil complexes, and iv) interact with metal complexes. Given our own contributions in the field, we place emphasis on peptide-templated labeling chemistry.

Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes

“…A distinct challenge is to perform chemical bioconjugation reactions in or on living cells. [1][2][3][4][5][6][7] Such reactions are useful for fluorescence labelling and imaging of proteins. Considering the naturally occurring functional group repertoire of biomolecules, it is difficult to site-specifically target a particular site or biomolecule without competing reactions at the multitude of other sites/biomolecules.…”

“…One solution to the specificity problem is provided by using enzymes that guide the chemistry to peptide recognition tags or enable self-modification reactions at the enzyme's specific microenvironment. [1][2][3][4][5][6][7] However, steric demand, the need for cofactors and carefully adjusted reaction conditions, or limited substrate scope can be reason for concern. 8,9 By contrast, chemical bioconjugation methods potentially offer tolerance to a wider scope of reaction conditions and substrates.…”

Orthogonal Peptide‐Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling

“…N-terminal labeling has been recently reviewed, highlighting advantages like fast reaction kinetics and versatility of coiled-coil-based systems compared to e. g. enzyme-based protein modification. [21] Our novel approach based on the Cys-P1/P3-tags adds only a small molecular weight increase (3 kDa) to the POI. Since the typical molecular weight of a GPCR is ~45 kDa, this is a minor modification compared to other common protein labeling techniques like the SNAP-tag (20 kDa) or Halo-tag (33 kDa), which minimizes the danger of impaired POI function and localization.…”

“…Since a negative impact of the Cys-P1/P3 modification on ET A R expression could not be excluded a priori, we characterized the ET A R-derived constructs concerning membrane localization and downstream signaling. The Cys-P1/P3-tag were introduced into the ET A R between S 20 and D 21 and wt-like G q signaling profiles were determined and efficient staining of membrane-embedded receptors was detected, indicating the presence of the SP. Some GPCR species display high sensitivity towards modification of their N-termini due to interference with membrane insertion and intracellular transport.…”

“…[15][16][17][18][19] Bio-orthogonal fluorescence labeling techniques are widely applied to study GPCR-GPCR interactions and the formation of receptor complexes in living cells. [20,21] Commonly, resonance energy transfer (RET) techniques like fluorescence and bioluminescence RET (FRET/BRET) are used to study the proximity of membrane-embedded GPCRs. [22][23][24] Often, RET-based methodologies rely on auto-fluorescent proteins, fused to the Cterminus of the protein of interest (POI), which prevents discrimination between membrane-embedded proteins from intracellular POI subpopulations.…”