Proteolysis at the cell surface is a critical mediator of developmental processes, cellular homeostasis, and tissue repair, but can play deleterious roles in disease states. By cleaving ECM components and cell adhesion receptors, cell surface proteolysis can have profound effects on cell–matrix and cell–cell interactions. Proteolysis at the cell surface also accounts for ectodomain shedding, the process of cleaving latent progrowth factors and cytokines to release active forms from cell membrane attachments.
Four classes of membrane-anchored cell surface proteases have been identified that participate in these cleavage events: membrane-type matrix metalloproteases (MT-MMPs),*
ectopeptidases, meprins, and ADAMs (a disintegrin and metalloprotease) (Stocker and Bode, 1995
; Bauvois, 2001
). Most of these cell surface proteases are type I integral membrane proteins that contain additional domains downstream of their protease domains (). Some of these nonproteolytic ectodomain elements have been shown to serve adhesive functions in vitro (Overall, 2002
). For example, the hemopexin domain of MT1-MMP, the cysteine-rich domain of dipeptidylpeptidase IV, and the disintegrin and/or cysteine-rich domain of Xenopus
ADAM13 have been shown, in in vitro biochemical experiments, to interact with ECM components such as collagen and fibronectin (De Meester et al., 1999
; Seiki, 1999
; Gaultier et al., 2002
). The disintegrin domains of several ADAMs have also been shown to support integrin-mediated cell adhesion (Takahashi et al., 2001
; Bridges et al., 2002
; Eto et al., 2002
). Very little is known about the role of the cysteine-rich domain in overall ADAM function. One recent in vitro study reported that the cysteine-rich domain of ADAM12 can interact with syndecans and mediate integrin-dependent cell spreading (Iba et al., 2000
). Although various in vitro binding partners for these putative adhesive domains have been identified, it is not known if, or how, this binding might contribute to substrate selection and proteolytic function in vivo. We are investigating these fundamental questions for ADAM proteases.
Figure 1. Schematic representation of cell surface proteases. Each is anchored in the membrane with a transmembrane domain or GPI linker. MT-MMPs and ectopeptidases come in both types, whereas meprins and ADAMs both possess transmembrane domains and cytoplasmic (more ...)
ADAM metalloproteases have been implicated in diverse developmental events such as fertilization, ECM remodeling, growth factor ectodomain shedding, and neurogenesis. ADAM proteases have a wide variety of substrates. They can degrade ECM components, shed cell-bound ectodomains to free growth factors and ligands from the cell surface, and cleave other integral membrane proteins (Blobel, 2000
; Primakoff and Myles, 2000
; Moss et al., 2001
; Kheradmand and Werb, 2002
). Through these mechanisms, ADAMs participate in cell migration, morphogenesis, tissue repair, and cell fate decisions. For the present study, we constructed chimeras between Xenopus
ADAM10 and -13 in order to assess the contributions of the downstream, nonproteolytic domains to overall ADAM protease function in vivo. Although similarly structured, ADAMs 10 and 13 have different roles in development.
ADAM13 is required for cranial neural crest cell migration, possibly by remodeling the fibronectin matrix en route (Alfandari et al., 2001
). In addition to causing alterations in cranial neural crest morphology and behavior, overexpression of transcripts encoding ADAM13 results in hyperplasia of the cement gland (Cousin et al., 2000
). The cement gland is the first ectodermally derived organ to differentiate in Xenopus
embryos. It arises in the anteriormost part of the embryo and marks the dorsal–ventral axis boundary. Cement gland induction requires a gradient of the growth factor bone morphogenetic protein-4 (BMP4) as well as counter expression of its inhibitors such as noggin, follistatin, and chordin. Retinoic acid, eFGF, and Xwnt8 are also required to make a cement gland (Sive and Bradley, 1996
). Many of these growth factors are first synthesized as membrane-tethered precursors and require extracellular proteolysis (shedding) to become active.
ADAM kuzbanian (kuz, also ADAM10) participates in axon extension through the ECM; kuz-null axons fail to form outgrowths (Fambrough et al., 1996
; Schimmelpfeng et al., 2001
). This failure of extension could be due to the matrix-degrading actions of kuz, or to its ability to bind and cleave ephrins (Ilan and Madri, 1999
). Loss of kuz function in fly embryos results in perturbation of Notch signaling, which affects neurogenesis (Pan and Rubin, 1997
; Lieber et al., 2002
). Preliminary work in Xenopus
suggests that ADAM10 plays a role in frog primary neurogenesis as well (Pan and Rubin, 1997
). X-ADAM10 mRNA is expressed maternally throughout the embryo and then later becomes restricted to a pan-neural expression pattern (Pan and Rubin, 1997
). As shown here, ADAM10 transcripts are also detected in the developing cement gland before its maturation. Although overexpression of wild-type ADAM10 message alters neural development, it has no affect on the genesis of the cement gland.
From our analyses of chimeras encoding domains of ADAMs 10 and 13, we find that the metalloprotease domain of either ADAM13 or ADAM10 is capable of inducing cement gland hyperplasia, but only if attached to the downstream adhesive domains of ADAM13. Further analyses indicate that the cysteine-rich domain of ADAM13 is absolutely essential to induce ectopic, expanded cement glands and, moreover, a disintegrin domain is required to support this behavior. Our data indicate that the cysteine-rich domains of ADAMs are important players in the biology of ADAM proteases in vivo. By extension, the nonproteolytic extracellular adhesive domains of other cell surface proteases (MT-MMPs, ectopeptidases, and meprins) may play similar roles.