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frequently found at sub-membranous localizations. Here, signal
transduction activities are commonly initiated through the dynamic
formation of protein complexes that are composed of multiple
PPIs [1]. For cell surface receptors, receptor tyrosine kinases
(RTKs) and G protein-coupled receptors (GPCRs) are intensively
studied protein classes, as deregulated signaling caused by these
receptors is implicated in various human diseases, such as cancer
and neurodevelopmental diseases [2, 3]. RTKs and GPCRs represent major drug targets, supporting their significant role in clinical
research and drug discovery [4]. Both the associations between
two proteins as well as the activities of RTKs and GPCRs can be
reliably studied in split biosensor assays (SBA, also called protein
complementation assays [5]). SBA are particularly suitable to monitor interactions of cell surface receptors and cytosolic proteins
with a sub-membranous localization as SBA facilitate an analysis in
the natural habitat of these proteins [6].
RTKs are single transmembrane receptors, with a total number
of 58 encoded in the human genome [3]. RTKs respond, with few
exceptions, to extracellular cues, and have an extra-cellular ligandbinding domain, a cytosolic tyrosine kinase domain, and a cytosolic
tail, which contains interaction motifs that establish, once phosphorylated through ligand-driven activation, docking sites for
cytosolic adapter proteins. Upon ligand binding, RTKs can
homodimerize or heterodimerize, leading to a conformational
change that transmits the signal into cytosol, where the kinase
domains phosphorylate target tyrosine residues in trans [7]. In
turn, Src homology 2 (SH2) domain- and phosphotyrosine binding (PTB) domain-containing adapters are recruited to phosphorylated docking sites, a process that can be robustly and precisely
monitored by SBA [8–10]. An established interaction between an
RTK and an adapter protein initiates downstream signaling activities, as these adaptors link RTK activation to downstream signal
transduction pathways, such as the MAP kinase or PI3K/AKT signaling cascades.
GPCRs are the largest class of receptors encoded in the human
genome, comprising more than 800 receptors in total [11]. Like
RTKs, deregulated GPCR signaling is strongly implicated in various human diseases. Strikingly, 30–40% of all marketed drugs target GPCRs, making them the largest class of druggable receptors
supporting their prominent role in drug discovery [4, 12]. GPCRs
are seven-transmembrane receptors with three intracellular and
three extracellular loops of varying length, an extracellular
N-terminus, and a cytoplasmic C-terminal tail, which can associate
with effector proteins in an activity-dependent manner [12].
Heterotrimeric G proteins are the major binding partners for
GPCRs and trigger defined pathway responses, such as cAMPmediated or Calcium-dependent pathway activities. Prolonged
activation of GPCR activation results in its desensitization, which
Jan P. Wintgens et al.
frequently found at sub-membranous localizations. Here, signal
transduction activities are commonly initiated through the dynamic
formation of protein complexes that are composed of multiple
PPIs [1]. For cell surface receptors, receptor tyrosine kinases
(RTKs) and G protein-coupled receptors (GPCRs) are intensively
studied protein classes, as deregulated signaling caused by these
receptors is implicated in various human diseases, such as cancer
and neurodevelopmental diseases [2, 3]. RTKs and GPCRs represent major drug targets, supporting their significant role in clinical
research and drug discovery [4]. Both the associations between
two proteins as well as the activities of RTKs and GPCRs can be
reliably studied in split biosensor assays (SBA, also called protein
complementation assays [5]). SBA are particularly suitable to monitor interactions of cell surface receptors and cytosolic proteins
with a sub-membranous localization as SBA facilitate an analysis in
the natural habitat of these proteins [6].
RTKs are single transmembrane receptors, with a total number
of 58 encoded in the human genome [3]. RTKs respond, with few
exceptions, to extracellular cues, and have an extra-cellular ligandbinding domain, a cytosolic tyrosine kinase domain, and a cytosolic
tail, which contains interaction motifs that establish, once phosphorylated through ligand-driven activation, docking sites for
cytosolic adapter proteins. Upon ligand binding, RTKs can
homodimerize or heterodimerize, leading to a conformational
change that transmits the signal into cytosol, where the kinase
domains phosphorylate target tyrosine residues in trans [7]. In
turn, Src homology 2 (SH2) domain- and phosphotyrosine binding (PTB) domain-containing adapters are recruited to phosphorylated docking sites, a process that can be robustly and precisely
monitored by SBA [8–10]. An established interaction between an
RTK and an adapter protein initiates downstream signaling activities, as these adaptors link RTK activation to downstream signal
transduction pathways, such as the MAP kinase or PI3K/AKT signaling cascades.
GPCRs are the largest class of receptors encoded in the human
genome, comprising more than 800 receptors in total [11]. Like
RTKs, deregulated GPCR signaling is strongly implicated in various human diseases. Strikingly, 30–40% of all marketed drugs target GPCRs, making them the largest class of druggable receptors
supporting their prominent role in drug discovery [4, 12]. GPCRs
are seven-transmembrane receptors with three intracellular and
three extracellular loops of varying length, an extracellular
N-terminus, and a cytoplasmic C-terminal tail, which can associate
with effector proteins in an activity-dependent manner [12].
Heterotrimeric G proteins are the major binding partners for
GPCRs and trigger defined pathway responses, such as cAMPmediated or Calcium-dependent pathway activities. Prolonged
activation of GPCR activation results in its desensitization, which
Jan P. Wintgens et al.