Abstract
We have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs). This system consists of at least nectin, a Ca(2+)-independent immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein, that connects nectin to the actin cytoskeleton. Nectin constitutes a family consisting of two members, nectin-1 and -2. We have isolated here a third member of the nectin family and named it nectin-3. Nectin-3 has three splicing variants, nectin-3alpha (biggest), -3beta (middle), and -3gamma (smallest). Like nectin-1 and -2, nectin-3alpha consists of three extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic region with the C-terminal consensus motif for binding to the PDZ domain. Nectin-3alpha formed a cis-homo-dimer and showed Ca(2+)-independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3alpha furthermore showed trans-hetero-interaction with nectin-1 or -2 but did not form a cis-hetero-dimer with nectin-1 or -2. Nectin-1 did not show trans-hetero-interaction with nectin-2. The affinity of trans-hetero-interaction of nectin-3alpha with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3alpha. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3alpha was colocalized with nectin-2 at cadherin-based AJs and interacted with afadin. These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs.
Highlights
We have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs)
These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs
Computer analysis of current protein data bases has revealed two molecules related to the nectin family members: PVR [27] and TAGE4 [28]
Summary
Molecular Cloning of the Mouse Nectin-3 cDNAs—The cDNAs of three mouse EST clones (AI1428160, AA492633, and AA497887) were amplified from a mouse brain cDNA (CLONTECH) by polymerase chain reaction. By use of the mixture of these cDNAs as a probe, a mouse cDNA library in uni-ZAP XR (Stratagene) was screened. Construction and Purification—Expression vectors were constructed in pCAGIPuro [14], pCAGIPuro-FLAG, pGEX-KG [20], pMal-C2 (New England Biolabs Inc.), and pFastBac1-Msp-Fc2 using standard molecular biology methods [21]. PCAGIPuro-FLAG was constructed by subcloning the insert encoding the preprotrypsin signal peptide and FLAG epitope of pFLAG-CMV1 (Eastman Kodak Co.) into pCAGIPuro. PFastBac1-Msp-Fc was constructed with a baculovirus transfer vector, pFastBac (Life Technologies, Inc.), to express a fusion protein with the N-terminal honeybee melittin signal peptide and the C-terminal IgG Fc as described.. Construction and Purification—Expression vectors were constructed in pCAGIPuro [14], pCAGIPuro-FLAG, pGEX-KG [20], pMal-C2 (New England Biolabs Inc.), and pFastBac1-Msp-Fc2 using standard molecular biology methods [21]. pCAGIPuro-FLAG was constructed by subcloning the insert encoding the preprotrypsin signal peptide and FLAG epitope of pFLAG-CMV1 (Eastman Kodak Co.) into pCAGIPuro. pFastBac1-Msp-Fc was constructed with a baculovirus transfer vector, pFastBac (Life Technologies, Inc.), to express a fusion protein with the N-terminal honeybee melittin signal peptide and the C-terminal IgG Fc as described. Constructs of mouse nectin-3 and rat afadin contained the following aa: pCAGIPuro-nectin-3␣, aa 1–549 (full length); pCAGI-
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