CPNA-1 was discovered independently by two strategies: (1) It was found as one of 4 new Pat mutants in an RNAi screen (Meissner et al. PLoS Genet. 2009). (2) It was identified in a two-hybrid screen as a binding partner for the N-terminal region (Immunoglobulin domains (Ig) 1-5) of the giant protein UNC-89 (obscurin). When CPNA-1 was used to test for interaction with two-hybrid clones covering all of UNC-89-B (8,081 residues), we detected binding only with the UNC-89 region containing Ig1-5. The minimal region of UNC-89 required for interaction is Ig1-3, and its interaction with CPNA-1 was confirmed by two biochemical methods. The N-terminus of CPNA-1 has a predicted transmembrane helix and near its C-terminus is a "copine domain", an ~180 residue long region with weak homology to the extracellular region of integrins, and unknown function. CPNA-1 is an "atypical" copine family protein as it has a copine domain but no C2 domains. We have found that C. elegans has 7 genes encoding proteins with copine domains: cpna-1 through cpna-5 are atypical, and nra-1 and gem-4 are typical. When tested by 2-hybrid against copine domains from each of these proteins, UNC-89 Ig1-5 interacted with only the copine domain from CPNA-1. CPNA-1 specific antibodies localize the protein to integrin adhesion complexes (M-lines and dense bodies) of nematode body wall muscle. We found that CPNA-1 binds to the M-line proteins UNC-89, LIM-9 (FHL), SCPL-1 (a CTD-type phosphatase), UNC-96, and a protein common to the M-line and dense body, PAT-6 (actopaxin). PAT-6 is a member of a 4-protein complex (including UNC-112 (Kindlin), PAT-4 (ILK) and UNC-97 (PINCH)) that bind the cytoplasmic tail of integrin. Previously it was shown that PAT-4 binds the C-terminal half of PAT-6 (Lin et al. Curr. Biol. 2003). We have shown that the N-terminal half of PAT-6 binds CPNA-1. A yeast 3-hybrid assay demonstrates a ternary complex can form between PAT-6, CPNA-1 and UNC-89. A null mutant for cpna-1, gk266, displays the typical Pat (Paralyzed arrested at two-fold) phenotype. The Pat embryonic lethal phenotype has been found in loss of function mutants of many components of muscle focal adhesion structures. By localizing previously characterized muscle adhesion complex proteins in cpna-1 mutant embryos, and localizing CPNA-1 in other Pat mutants, we have placed CPNA-1 in the M-line/dense body assembly pathway of embryonic muscle: CPNA-1 lies between PAT-6 and MYO-3. We hypothesize that PAT-6 recruits CPNA-1, and in turn, CPNA-1 recruits additional proteins (UNC-89, LIM-9, SCPL-1, UNC-96) to muscle focal adhesions.

Affiliations:
- Dept Zoology, Univ British Columbia, Vancouver, BC
- Dept Pathology, Emory Univ, Atlanta, GA.