Protein Chemistry on the Surface of Living Cells

Johnsson, Nils; George, Nathalie; Johnsson, Kai

doi:10.1002/cbic.200400290

Cited by 51 publications

(41 citation statements)

References 41 publications

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“…1). The chosen tags belong to the ACP and PCP families [3], [9] and bear a serine residue as the site of covalent transfer of the CoA PP arm by PPTase enzymes. In all experiments presented here, the CoA PP arm is substituted with biotin so that we achieve site-specific biotinylation of (pro)NGF and its receptors.…”

Section: Discussionmentioning

confidence: 99%

“…We previously demonstrated that the insertion of the acyl carrier protein (ACP) tag [3] at the extracellular domain of TrkA makes it possible to specifically label the receptor at the cell surface when the construct is transfected in living cells [4], [5]. The ACP tag belongs to a family of protein and peptide tags, which can be covalently conjugated to virtually any small-probe substituted phosphopantetheinyl (PP) arm of Coenzyme A (CoA) substrate by post-translational modification enzymes named PP transferases (PPTases) [6].…”

Section: Introductionmentioning

confidence: 99%

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Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

et al. 2014

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We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

et al. 2014

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“…[30] Avidin-functionalized semiconductor quantum dots and colloidal gold or latex nanoparticles could also be attached to ACP-modified membrane proteins with biotinylated CoA. [31] These would have the advantage of no or little photobleaching, but have the drawback of large sizes, intrinsic multivalency, and "stickiness". Depending on the preparation of quantum dots, frequent long dark states can render extended periods of tracking difficult.…”

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confidence: 99%

Post‐translational Covalent Labeling Reveals Heterogeneous Mobility of Individual G Protein‐Coupled Receptors in Living Cells

et al. 2006

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“…We used TIRF microscopy and SPT together with the ACP-TrkA construct, which allows the selective labelling and imaging of single receptor molecules translocated to the plasma membrane (Callegari et al, 2012). We used two different receptor labelling strategies exploiting the versatility of the ACP tag (George et al, 2004;Johnsson et al, 2005). In one case, we biotinylated ACP-TrkA using a CoAbiotin substrate and coupled them to S-Qdots; in the other, we covalently conjugated ACP-TrkA to a CoA derivative of the Atto633 synthetic fluorophore.…”

Section: Discussionmentioning

confidence: 99%

Ligand signature in the membrane dynamics of single TrkA receptor molecules

Marchetti

Callegari

Luin

et al. 2013

Journal of Cell Science

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SummaryThe neurotrophin receptor TrkA (also known as NTRK1) is known to be crucially involved in several physio-pathological processes. However, a clear description of the early steps of ligand-induced TrkA responses at the cell plasma membrane is missing. We have exploited single particle tracking and TIRF microscopy to study TrkA membrane lateral mobility and changes of oligomerization state upon binding of diverse TrkA agonists (NGF, NGF R100E HSANV mutant, proNGF and NT-3). We show that, in the absence of ligands, most of the TrkA receptors are fast moving monomers characterized by an average diffusion coefficient of 0.47 mm 2 /second; about 20% of TrkA molecules move at least an order of magnitude slower and around 4% are almost immobile within regions of about 0.6 mm diameter. Ligand binding results in increased slow and/or immobile populations over the fast one, slowing down of non-immobile trajectories and reduction of confinement areas, observations that are consistent with the formation of receptor dimeric and oligomeric states. We demonstrate that the extent of TrkA lateral mobility modification is strictly ligand dependent and that each ligand promotes distinct trajectory patterns of TrkA receptors at the cell membrane (ligand 'fingerprinting' effect). This ligand signature of receptor dynamics results from a differential combination of receptor-binding affinity, intracellular effectors recruited in the signalling platforms and formation of signalling and/or recycling endosome precursors. Thus, our data uncover a close correlation between the initial receptor membrane dynamics triggered upon binding and the specific biological outcomes induced by different ligands for the same receptor.

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Protein Chemistry on the Surface of Living Cells

Cited by 51 publications

References 41 publications

Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

Post‐translational Covalent Labeling Reveals Heterogeneous Mobility of Individual G Protein‐Coupled Receptors in Living Cells

Ligand signature in the membrane dynamics of single TrkA receptor molecules

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