Cytoplasmic domain of protocadherin‐α enhances homophilic interactions and recognizes cytoskeletal elements

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Abstract
Cell adhesion molecules of the protocadherin-α (pcdh-α), -β, and -γ families have been proposed to be synaptic specifiers. Pcdh-α and -γ family members localize in part to synapses, and deletion of all pcdh-γs in mouse affects synaptogenesis. Little is known, however, about the binding specificities and intracellular signaling of protocadherins. Using heterologous expression of tagged constructs, immunostaining, and biotinylation of surface components followed by Western blots we demonstrate that pcdh-αs undergo homophilic interactions that are significantly enhanced by the cytoplasmic domain. Pcdh-αs cloned from chick ciliary ganglion have one of two cytoplasmic constant regions (A- and B-types). Screening a yeast two-hybrid library of ciliary ganglion cDNA with the A-type domain yielded a fragment of neurofilament M (NFM); screening with B-type domain yielded a fragment of the actin-bundling protein fascin. Cotransfection of HEK cells with the constructs indicated that the NFM and A-type fragments codistributed as did the fascin and B-type fragments, and the latter could be coimmunoprecipitated. Antibody-induced clustering of full-length pcdh-αs on the surface of transfected HEK cells induced coclustering of the interacting NFM fragment. Native full-length NFM in tissue extracts bound specifically to the A-type domain on beads, while native full-length fascin in tissue extracts specifically coimmunoprecipitated with pcdh-α. Immunostaining neurons demonstrated codistribution of full-length pcdh-α with both NFM and actin filaments. These findings suggest cytoskeletal links for pcdh-αs and identify candidate targets. They also demonstrate homophilic interactions for pcdh-αs as described for classical cadherins. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006