Trends in Pharmacological Sciences
ReviewEngineering receptors activated solely by synthetic ligands (RASSLs)
Section snippets
RASSL construction
For our first RASSL, we modified the KOR. This receptor responds to endogenous peptides and signals via the well-characterized Gi pathway. Because of the importance of this receptor family for pain modulation, the pharmaceutical industry has developed many high-affinity opioid receptor agonists. Small-molecule ligands of the KOR are structurally distinct from the endogenous peptide ligands, including dynorphin 11, 12. Unlike mu or delta opioid receptor agonists, KOR agonists are nonaddictive 13.
Control of RASSL expression in vivo
The expression of RASSLs in specific tissues of whole animals is a potentially valuable tool for studying both normal and pathological signaling cascades. To take full advantage of the signaling control offered by a RASSL, it is also important to control the timing and location of RASSL expression in vivo. To achieve this, we have used the tetracycline-controlled inducible expression system (tet system) developed by Gossen and Bujard 20. Briefly, transgene expression is driven by a minimal
Controlling RASSL signaling in vivo
When the receptor is expressed, it should be functionally silent until the administration of spiradoline stimulates the RASSL and activates signaling pathways rapidly and specifically. Gi signaling in the heart has been shown to reduce heart rate 26. Therefore, when Ro1 is expressed in the heart (using the α-myosin heavy chain promoter), receptor activation can be studied by simply measuring changes in heart rate. Wild-type animals have few KORs in the heart and therefore show no change in
Detecting RASSLs in vivo
For many experiments that use RASSLs to study the role of Gi signaling in vivo, it is essential to pinpoint the receptor localization, sometimes down to the subcellular level. To this end, we have developed several tagged RASSLs*.
Small epitope tags have been added to the N-terminus of the RASSL protein for subsequent detection with antibody staining. Common tags such as hemagglutinin (HA)
Applications of RASSL technology
Acute activation of RASSLs expressed in discrete tissues or cell types will allow researchers to probe the role of receptor-mediated Gi signaling in normal cell function. Correspondingly, extended activation of RASSLs can be used to explore disease models related to hyperactive G-protein signaling.
Tissue-specific expression and activation of RASSLs
To date, RASSLs have been expressed in multiple tissues of transgenic mice (Table 1), including heart, liver, salivary glands, smooth muscle, adipose tissue and specific brain regions. Ro1-mediated Gi activation has been shown to slow heart rate 5. RASSLs are being used to study the effects of G proteins on smooth muscle contractility, and mouse lines that express RASSLs in specific brain regions are being used to study how activation of Gi signaling in specific brain nuclei can affect
Future directions: new RASSLs
Existing RASSLs will allow study of the effects of Gi-mediated signaling in specific tissues under specific circumstances. In the future, RASSLs could be made to control all the major G-protein signaling pathways, individually or in combination (Gs, Gi, Gq, G12, G13). These new RASSLs can be developed using many of the same principles used to develop the existing Gi-coupled RASSLs. The ideal RASSL would have orally available, nanomolar affinity agonists and antagonists that are safe for
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