Linking molecular affinity and cellular specificity in cadherin-mediated adhesion

Abstract

Many cell-cell adhesive events are mediated by the dimerization of cadherin proteins presented on apposing cell surfaces. Cadherin-mediated processes play a central role in the sorting of cells into separate tissues in vivo, but in vitro assays aimed at mimicking this behavior have yielded inconclusive results. In some cases, cells that express different cadherins exhibit homotypic cell sorting, forming separate cell aggregates, whereas in other cases, intermixed aggregates are formed. A third pattern is observed for mixtures of cells expressing either N- or E-cadherin, which form distinct homotypic aggregates that adhere to one another through a heterotypic interface. The molecular basis of cadherin-mediated cell patterning phenomena is poorly understood, in part because the relationship between cellular adhesive specificity and intermolecular binding free energies has not been established. To clarify this issue, we have measured the dimerization affinities of N-cadherin and E-cadherin. These proteins are similar in sequence and structure, yet are able to mediate homotypic cell patterning behavior in a variety of tissues. N-cadherin is found to form homodimers with higher affinity than does E-cadherin and, unexpectedly, the N/E-cadherin heterophilic binding affinity is intermediate in strength between the 2 homophilic affinities. We can account for observed cell aggregation behaviors by using a theoretical framework that establishes a connection between molecular affinities and cell-cell adhesive specificity. Our results illustrate how graded differences between different homophilic and heterophilic cadherin dimerizaton affinities can result in homotypic cell patterning and, more generally, show how proteins that are closely related can, nevertheless, be responsible for highly specific cellular adhesive behavior.

Fig. 1.

Structure of cadherin molecules. (A) The structure of the adhesive dimeric complex formed by ectodomains of type I C-cadherin (7) is shown in molecular surface representation. Note that the adhesive interface is encompassed entirely within the EC1 domain. The red boxed region includes the interacting EC1–2 regions corresponding to the cadherin constructs used in this study. (B) Expanded ribbon diagram view of the strand-exchanged adhesive interface between the N-terminal EC1 domains of the type I E-cadherin (9). The side chains of the Trp anchor residues are shown. (C) A ribbon diagram view of the strand-exchanged adhesive interface between the N-terminal EC1 domains of the type II cadherin 8 (15). The side chains of the Trp-2 and Trp-4 anchor residues are indicated.

Fig. 2.

Mixing cell aggregation assays with cadherin-expressing CHO cells. Mixing aggregation assays using 2 identical CHO cell lines expressing the same cadherin—(A) N-cadherin with N-cadherin, (B) E-cadherin with E-cadherin, and (C) cadherin-6b with cadherin-6b—result in aggregates composed of an interspersed mixture of each cell line. (D) Dissociated cells expressing E- and N-cadherins form separate homotypic aggregates that adhere to one another. In contrast, cells that express type II cadherin-6b form aggregates that do not adhere to cells expressing either E-cadherin (E) or N-cadherin (F). (Magnification: 20×.)

Fig. 3.

SPR binding experiments using N- and E-cadherins. (A) Biotinylated cadherin of a given type (ligand, shown in blue) was tethered over a neutravidin-coated sensor chip. Another cadherin of a given type (analyte, shown in red) was injected independently over the surface. The interaction between an analyte monomer (red) and the ligand monomer (blue) produces a binding signal. (B) N-cadherin (blue traces) and E-cadherin (red traces) were injected over a surface containing N-cadherin at 30 μM and 17 μM, respectively, which correspond to 13.1 μM free monomer. Each sample was injected in duplicate. N-cadherin W2A (orange traces) and E-cadherin W2A (green traces) mutants, which were also injected at the same monomer concentrations, show no binding. Inset shows an overlay of buffer blank injections performed throughout the experiment over the same surface. (C) During the same experiment, N- and E-cadherins were also injected over a surface containing E-cadherin, under the same conditions as described in B. Mouse cadherin-6 (purple traces) was injected at 121 μM (effective monomer concentration, 13.1 μM) over the same E-cadherin (D) and N-cadherin (E) surfaces. Black traces represent buffer blanks that were performed throughout the experiment. (F) To confirm that lack of binding was not due to an inactive protein, binding of cadherin-6 was tested against a cadherin-6 surface.