Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling

Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression.These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic Gi-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute Gi signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo.The specificity, diversity, and physiological importance of G protein-coupled receptors (GPCR) have made these receptors excellent drug targets. It is becoming clear that the regulation of the GPCR itself – its location, stability, and signal duration – is a key component of the signaling process [1,2] The length of a GPCR signal can be modulated by receptor desensitization (decrease in receptor responsiveness) and receptor internalization (trafficking of receptors to endocytotic vesicles). The cell can also respond to prolonged activation by upregulating compensatory pathways. For example, prolonged signaling through a Gi-coupled receptor inhibits adenylyl cyclase (AC), while paradoxically increasing the ability of the Gs-coupled pathway to stimulate AC, a phenomenon known as AC superactivation [3]. Such regulatory mechanisms may con