Existing pharmacotherapies
acting on the opioid receptor system
have been extensively used to treat chronic pain and addictive disorders.
Nevertheless, the adverse side effects associated with opioid therapy
underscore the need for concerted measures to develop safer analgesics.
A promising avenue of research stems from the characterization of
a sodium-dependent allosteric regulation site housed within the delta-opioid
receptor and several other G protein-coupled receptors (GPCRs), thereby
revealing the presence of a cluster of sodium and water molecules
lodged in a cavity thought to be present only in the inactive conformation
of the receptor. Studies into the structure–function relationship
of said pocket demonstrated its critical involvement in the functional
control of GPCR signaling. While the sodium pocket has been proposed
to be present in the majority of class A GPCRs, the shape of this
allosteric cavity appears to have significant structural variation
among crystallographically solved GPCRs, making this site optimal
for the design of new allosteric modulators that will be selective
for opioid receptors. The size of the sodium pocket supports the accommodation
of small molecules, and it has been speculated that promiscuous amiloride
and 5′-substituted amiloride-related derivatives could target
this cavity within many GPCRs, including opioid receptors. Using pharmacological
approaches, we have described the selectivities of 5′-substituted
amiloride-related derivatives, as well as the hitherto undescribed
activity of the NHE1 inhibitor zoniporide toward class A GPCRs. Our
investigations into the structural features of the delta-opioid receptor
and its ensuing signaling activities suggest a bitopic mode of overlapping
interactions involving the orthosteric site and the juxtaposed Na
+
pocket, but only at the active or partially active opioid
receptor.
Protein-protein interactions (PPIs) form the underpinnings of any cellular signaling network. PPIs are highly dynamic processes and often, cell-based assays can be essential for their study as they closely mimic the biological intricacies of cellular environments. Since no sole platform can perform all needed experiments to gain a thoroughly comprehensive understanding into these processes, developing a versatile toolkit is much needed to address this longstanding gap. The use of small peptide tags, such as the V5-tag, has been extensively used in biological and biomedical research, including labeling the C-termini of one of the largest human genome-wide open-reading frame collections. However, these small peptide tags have been primarily used in vitro and lack the in vivo traceability and functionality of larger specialized tags. In this study, we combined structural studies and computer-aided maturation to generate an intracellular nanobody, interacting with the V5-tag. Suitable for assays commonly used to study protein-protein interactions, our nanobody has been applied herein to interrogate G protein-coupled receptor signalling. This novel serviceable intrabody is the cornerstone of a multipurpose intracellular nanobody-based biosensors toolkit, named iBodyV5, which will be available for the scientific community at large.
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