Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.

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Alzheimer disease (AD) is characterized by deposition of amyloid-β, tau, and other specific proteins that accumulate in the brain in detergent-insoluble complexes. AD also involves glutamatergic neurotransmitter system disturbances. Excitatory amino acid transporter 2 (EAAT2) is the dominant glutamate transporter in cerebral cortex and hippocampus. We investigated whether accumulation of detergent-insoluble EAAT2 is related to cognitive impairment and neuropathologic changes in AD by quantifying detergent-insoluble EAAT2 levels in hippocampus and frontal cortex of cognitively normal patients, patients with clinical dementia rating (CDR) = 0.5 (mildly impaired), and AD patients. Parkinson disease (PD) patients served as neurodegenerative disease controls. We found that Triton X-100-insoluble EAAT2 levels were significantly increased in patients with AD compared to controls, while Triton X-100-insoluble EAAT2 levels in CDR = 0.5 patients were intermediately elevated between control and AD subjects. Detergent-insolubility of Presenilin-1, a structurally similar protein, did not differ among the groups, thus arguing EAAT2 detergent-insolubility was not due to nonspecific cellular injury. These findings demonstrate that detergent-insoluble EAAT2 accumulation is a progressive biochemical lesion that correlates with cognitive impairment and neuropathologic changes in AD. These findings lend further support to the idea that dysregulation of the glutamatergic system may play a significant role in AD pathogenesis.