Pertussis toxin induces parallel loss of neuropeptide Y Y1 receptor dimers and Gi alpha subunit function in CHO cells

Area of Science:

• Cellular and Molecular Pharmacology
• G Protein-Coupled Receptor Signaling
• Neuroendocrinology

Background:

• Neuropeptide Y Y(1) receptors play crucial roles in various physiological processes.
• G protein-coupled receptors (GPCRs) mediate cellular responses through interactions with intracellular G proteins.
• Pertussis toxin is a tool used to selectively inhibit G(i)alpha subunits of heterotrimeric G proteins.

Purpose of the Study:

• To investigate the effect of pertussis toxin-mediated G(i)alpha subunit inhibition on human neuropeptide Y Y(1) receptor expression and function.
• To explore the potential co-regulation and protective mechanisms between Y(1) receptors and G(i)alpha subunits.

Main Methods:

• Expression of human neuropeptide Y Y(1) receptors in Chinese hamster ovary (CHO) cells.
• Treatment with pertussis toxin to inhibit G(i)alpha subunits.
• Assays for adenylyl cyclase activity and GTPgammaS binding to assess receptor-G protein coupling.
• Treatment with ammonium chloride to investigate the role of acid proteinases.
• Analysis of receptor surface expression and agonist-induced internalization.

Main Results:

• Pertussis toxin treatment significantly reduced Y(1) receptor levels and abolished agonist-mediated inhibition of adenylyl cyclase and GTPgammaS binding.
• Ammonium chloride treatment attenuated the loss of both receptor and G(i)alpha subunit function, suggesting co-protection.
• While surface receptor levels decreased, agonist-induced internalization was reduced by approximately 70%.
• A preserved fraction of Y(1) receptors was found to be coupled to G(q)alpha subunits.
• Persistent, pertussis toxin-insensitive internalization suggests alternative signaling pathways or receptor rescue.

Conclusions:

• Inactivation of G(i)alpha subunits by pertussis toxin leads to a substantial loss of functional Y(1) receptors, likely involving receptor oligomers.
• A co-protective mechanism exists between active G(i)alpha subunits and Y(1) receptors, potentially mediated by acid proteinases.
• The findings highlight the complex regulation of Y(1) receptor signaling and internalization, with potential implications for cells in neuropeptide Y-rich environments.