Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor

The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sj?gren’s syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R^2nd mAbs) and analyzed their function by Ca²⁺-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R^2nd mAbs suppressed Ca²⁺-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.