Muscarinic acetylcholine receptors (MRs), a family of five G protein-coupled receptors (GPCRs), play an essential role in the regulation of mammalian physiology. In the brain, MR-mediated neurotransmission is required for the control of movement and motivated behavior by the basal ganglia, and MR dysfunction may contribute to schizophrenia, Alzheimer's disease, and motor disorders. Functional studies of the muscarinic receptors have been hampered by a lack of selective pharmacology, poor receptor immunoreactivity and a wide, overlapping pattern of expression. MRs are characterized by the presence of a large third intracellular loop domain (i3), the sequence of which is divergent between MR subtypes. The i3 is known to determine signaling and trafficking characteristics of GPCRs by binding to defined subsets of regulatory and effector proteins. In an effort to discover novel, subtype-specific muscarinic receptor regulatory mechanisms, we performed yeast two-hybrid proteinprotein interaction screens with the five MR i3 regions. An interaction between M5 and the Arf GAP protein AGAP1 was detected, and was observed to be specific to the M5 subtype. This interaction was confirmed in vitro, and was shown to mediate the binding of the AP-3 adaptor complex to the M5 i3. Immunocytochemical and live cell imaging of primary rat hippocampal neurons revealed co-localization of M5 and AGAP1- or AP-3- positive vesicles after treatment with a muscarinic agonist. Activity-induced receptor trafficking studies demonstrated that interaction with AGAP1 and activity of AP-3 were required for the endocytic recycling of M5 in neurons, the lack of which resulted in downregulation of cell surface receptor density. M5 has been shown to be expressed in the dopaminergic neurons of the ventral midbrain and to function in the presynaptic modulation of dopamine release in the striatum. Results from dopamine release studies suggest that the abrogation of AGAP1-mediated recycling decreases the magnitude of presynaptic M5-mediated release potentiation. Our study demonstrates a novel, neuronspecific trafficking function for AGAP1 and AP-3, and suggests the presence of a previously unknown receptor recycling pathway that may underlie mechanisms of sustained sensitivity of GPCRs.

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy