The abundance of the cell adhesion molecule N-cadherin is one factor that contributes to dendritic spine plasticity. When N-cadherin activity is compromised, spine number is decreased. Yasuda et al. found that conditions that stimulate excitatory synapses, such as maximal electroconvulsive seizure in mice or application of glutamate or agents that elevate the concentration of adenosine 3′,5′-monophosphate (cAMP) to cultured hippocampal neurons, increased the abundance of arcadlin, a protocadherin protein that was recruited to excitatory dendrites. Furthermore, arcadlin coimmunoprecipitated with N-cadherin from rat hippocampi after stimulation, as well as from transfected cultured cells. Cultured cells transfected with arcadlin and N-cadherin exhibited diminished homophilic adhesiveness. Stimulated hippocampal cultures exhibited decreased abundance of N-cadherin at the cell surface (based on a surface biotinylation assay and immunofluorescent analysis). In neurons cultured from acad–/– mice, increases in cAMP concentration did not decrease N-cadherin surface abundance. A yeast two-hybrid screen identified a kinase that the authors named TAO2β, which appears to be an alternative splice variant of TAO2, as a protein that interacted with arcadlin. This interaction was confirmed by coimmunoprecipitation of the two proteins from hippocampi of mice after maximal electroconvulsive seizure and in colocalization studies in transfected cells. TAO2 is a mitogen-activated protein kinase kinase kinase (MAPKKK), and when transfected cells or primary cultures of rat hippocampal neurons were treated with the arcadlin extracellular domain (Acad-EC) to ligate the arcadlin proteins, phosphorylated mitogen-activated protein kinase kinase 3 (MEK3) and p38 MAPK were detected, suggesting that TAO2β is also a p38 MAPKKK. The abundance of arcadlin at the cell surface, as well as that of N-cadherin, was decreased in response to Acad-EC through a mechanism that required TAO2β and p38 activity. TAO2 could not substitute for TAO2β in triggering arcadlin endocytosis, yet endocytosis required p38, suggesting a possible feedback mechanism depending on TAO2β. Indeed, in vitro phosphorylation assays confirmed that TAO2β was a substrate for p38, and, in cells transfected with mutants that lacked the phosphorylation site, the importance of this phosphorylation for triggering endocytosis of arcadlin was confirmed. Finally, the increase in spine density seen in cultured neurons from acad–/– mice was reversed by knockdown of N-cadherin. Thus, arcadlin appears to link the endocytosis of N-cadherin to excitatory stimuli through a MAPK pathway.
S. Yasuda, H. Tanaka, H. Sugiura, K. Okamura, T. Sakaguchi, U. Tran, T. Takemiya, A. Mizoguchi, Y. Yagita, T. Sakurai, E. M. De Robertis, Activity-induced protocadherin arcadlin regulates dendritic spine number by triggering N-cadherin endocytosis via TAO2β and p38 MAP kinases. Neuron 56, 456-471 (2007). [PubMed]