GPCR modifying proteins

Andrea Kliewer,

Friedrich-Schiller University Jena, Germany

Insights into GRK and β-arrestin mediated μ-opioid receptor regulation in vivo

Opioids, such as morphine and fentanyl, acting through the µ-opioid receptor (MOP) and are the cornerstone therapy for treatment of acute and chronic pain. However, their clinical use is limited by severe adverse side effects such as respiratory depression, constipation, dependence and development of tolerance after chronic administration. Opioid analgesic misuse and overdose death are major public health concerns worldwide. However, we still do not have an clear mechanistical understanding of MOP-induced brain effects, especially those leading to adverse side effects. This limits the development of novel pain-relieving drugs with reduced side effects.

Recently, by using phosphorylation-deficient mutant knockin mice (10S/T-A) that are unable to recruit regulatory β-arrestin proteins, we have identified carboxyl-terminal multi-site phosphorylation as the key towards acute MOP desensitization and long-term tolerance. Strikingly, respiratory depression, constipation, and opioid addiction are unchanged or exacerbated in the knock-in mice.

Since MOP desensitization is highly regulated by GRKs and β-arrestins we have generated different new MOP mouse models where can be investigated how these intracellular proteins are influencing the development of opioid-mediated side effects.

Brian Muntean,

Augusta University, Georgia, USA

Emerging role of KCTD proteins in striatal cAMP signaling

Many GPCRs/G proteins couple to cAMP signaling, establishing a highly sensitive system fine-tuned by additional regulators and modifiers, which adjusts neuronal activity. Data over the years shows that mutations in transducers and regulators of the cAMP cascade, many of which are highly expressed in the striatum, are connected to hyperkinetic movement disorders. However, the complexity of cAMP regulation in the striatum and the large number of players with pivotal roles that have yet to be fully elucidated has slowed progress towards more effective therapeutic strategies.

Using a multidisciplinary approach, we identified an essential role for members of the Potassium Channel Tetramerization Domain (KCTD) family in decoding neuromodulatory signals in striatal neurons. We find that KCTD’s dynamically adjust availability of GPCR machinery thus driving signal regulation through diverse mechanisms. First, cAMP is regulated by KCTD5-mediated modulation of zinc influx through the Zip 14 transporter. In addition, KCTD5 also controls Gβγ-mediated cAMP production. Finally, disruption of striatal cAMP in KCTD5 haploinsufficient mice leads to motor deficits that can be reversed by zinc chelation.

Together, our results show KCTD exhibit manifold regulation in tuning cAMP signals by acting as molecular switches setting the compositional landscape of critical signaling determinants along the GPCR-cAMP axis.

Katarina Nemec,

Max Delbrück Center for Molecular Medicine, Berlin, Germany

RAMP gets a new role - modulation of activation dynamics

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR, and an essential modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function.

Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we present that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Lastly, by employing homology modeling, we describe the putative structural molecular basis underlying our functional findings.

I will demonstrate how these data add to a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for precise modulation of GPCR function and advanced drug design.