News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
Regulation, Function, and Signaling Mechanisms of Cell Adhesion Molecules
The fundamental processes of embryogenesis have evolved to translate the linear
genetic code reproducibly a into 3-dimensional organism. Early in development,
dividing cells derived from the fertilized egg adhere into specific aggregates.
The gene program of each aggregate is then altered, moving the cells down differentiation
pathways leading to specific tissues and organs. My colleagues and I have identified
and characterized adhesion proteins on the surfaces of cells that allow aggregation,
and we have shown that different tissues have different combinations of these
cell adhesion molecules or CAMs. One of our objectives is to describe the signals
that regulate the expression of gene programs when cells aggregate via specific
In addition to forming tissues and organs, development must proceed in such
a way that each collective or aggregate of cells is in its proper place in the
overall body plan. Transcription factors such as the protein products of genes
called Hox and Pax are among the regulators of this process. These
factors act by turning sets of other genes on or off at critical places along
the axis of the embryo. We showed that CAM genes are targets for proteins encoded
by Hox and Pax, indicating that regulation of CAM gene expression
may play a role in tissue morphogenesis in specific regions of the embryo. Our
second major goal is to define the cellular processes and cellular components
in addition to products of Hox and Pax that are responsible for
changing the expression of CAM genes during developmental processes.
Genes are controlled through regions of the DNA called promoters, and transcription
factors can bind to specific parts of the promoter DNA to activate or repress
gene expression. In previous studies, we found that several proteins encoded
by Hox and Pax alter the expression of a number of CAM genes. We
discovered that in addition to being regulated by Hox and Pax proteins, 2 related
CAMs restricted to the nervous system, Ng-CAM and L1, contain a 21-bp negative
regulatory element known as the neural restrictive silencer element. This element
and its binding factor, REST/NRSF, are responsible for silencing the expression
of these CAMs in nonneural tissues.
In our most recent studies, we identified an important positive regulatory
element, the HPD, within the gene for L1 that binds to both homeobox and Pax
proteins. The HPD carries out a number of important regulatory functions for
the gene. It is required for binding to and activation of the L1 promoter by
the transcription factor Pax-6, is necessary for the proper pattern of L1 expression
in the forebrain and midbrain, and is critical for the response of the gene to
bone morphogenetic proteins. Bone morphogenetic proteins are signaling proteins
secreted by cells during embryonic induction and are important regulators of
morphogenesis in both neural and nonneural tissues. We are investigating the
contexts by which these proteins regulate the expression of CAMs and are identifying
the DNA elements and transcription factors that participate in these responses.
In previous studies, we showed that the neural CAM (N-CAM) inhibits astrocyte
proliferation in vitro and in vivo after a lesion. Furthermore, N-CAM binding
affected mitogen-activated protein kinase activity in astrocytes and altered
gene expression in both astrocytes and neurons. These functions of N-CAM depended
on activation of the glucocorticoid receptor, a transcription factor. These findings
led us to consider effects of N-CAM on other transcription factors, such as AP-1
and NF-κB, that are also influenced by the glucocorticoid receptor.
Recently, we analyzed DNA binding of and transcriptional activation by NF-κB
after N-CAM binding to the cell surface. Addition of purified N-CAM, the recombinant
third immunoglobulin (Ig) domain of N-CAM, or N-CAM antibodies to neonatal rat
forebrain astrocytes or to cerebellar granule neurons increased NF-κBDNA
binding activity in nuclear extracts, as measured by electrophoretic mobility
shift assays. NF-κBmediated transcription was increased after N-CAM
binding to astrocytes and neurons, as shown by the activation of 2 different
luciferase reporter constructs containing NF-κB binding sites. N-CAM binding
also resulted in degradation of IκB-α protein, which inhibits the
translocation of NF-κB into the nucleus, and a subsequent increase in the
levels of IκB-α mRNA and protein.
These results indicate that N-CAM homophilic binding at the cell membrane
leads to activation of NF-κB binding to DNA and transcriptional activation
in both neurons and astrocytes. Activation of NF-κB, however, did not influence
the ability of N-CAM to inhibit astrocyte proliferation. These observations together
support the hypothesis that N-CAM binding activates multiple pathways leading
to changes in gene expression in astrocytes and neurons. Ongoing studies are
aimed at elucidating intracellular signaling pathways and genes that are regulated
by cell adhesion. Moreover, we are pursuing the hypothesis, based on our earlier
work, that N-CAM synthesis is regulated in part by synaptic activity.
Despite extensive studies of CAMs, little is known about the mechanism of
binding of these molecules or how this binding leads to intracellular signals
that eventually lead to changes in gene expression. The goal of our studies is
to describe the molecular events involved in these processes, with a focus on
N-CAM. The mechanism of homophilic N-CAM binding is being investigated initially
by looking for specific interactions between the extracellular Ig domains (Ig
IIg V) of the protein. In collaborative studies with J. Dyson and P. Wright,
The Scripps Research Institute, the distal Ig domain (Ig I), was uniformly labeled
with carbon 13 and nitrogen 15, and the backbone and side chain assignments were
determined on the basis of triple-resonance nuclear magnetic resonance experiments.
Knowledge of the assignments of Ig I allowed us to probe potential interactions
with other domains through chemical-shift perturbations induced by protein-protein
binding. Using this method, we detected a weak but specific interaction between
Ig I and Ig II, centered on a hydrophobic surface on Ig I. Under similar experimental
conditions, Ig I did not bind to itself and did not interact with Ig III.
Further support for an interaction between the first and second Ig domains
was obtained from biophysical studies in which the protein corresponding to these
2 domains was expressed as a single polypeptide chain. In analytical ultracentrifugation
experiments performed by H. Lashuel and J. Kelly, the Skaggs Institute, the Ig
IIg II concatemer dimerized with a significantly stronger association constant
than that of the individual Ig I and Ig II domains, consistent with an antiparallel
orientation of the molecules. These results suggest that an interaction can occur
between these domains and provide new views on how an N-CAM molecule on one cell
may interact with an N-CAM molecule on the same cell or on an apposing cell.
To assess the structural features of the cytoplasmic domain that are involved
in N-CAM signaling events, we transfected cells with constructs that express
the native and mutated forms of the cytoplasmic domain. Initial studies focused
on the 4 cysteines immediately adjacent to the internal face of the plasma membrane
that were assumed to be the sites where N-CAM is acylated with fatty acids. Site-directed
mutagenesis verified that these are the sites of palmitoylation and indicated
that the cysteines are differentially acylated. The results also indicated that
the fatty acids help direct the protein to the membrane and help anchor it there.
The cytoplasmic domain without the transmembrane region was directed to the membrane
fraction but only if at least 2 of the cysteines were present.
These results suggest that fatty acid acylation influences the association
of N-CAM with the membrane and may be important in localization of the protein
or in organizing the structure of the cytoplasmic segment. These studies are
being extended to show that the cytoplasmic domain can have a dominant effect
on N-CAM signaling and to assess the role of the various phosphorylation sites
on N-CAM function. For the latter studies, the cytoplasmic domain is expressed
with the membrane-spanning region to ensure proper orientation in the cell and
to maximize expression.
Cell differentiation during development can be influenced by controlling translation
of mRNA and by gene transcription. In earlier studies, we observed that large
numbers of eukaryotic mRNAs contain sequences that resemble segments contained
within the 18S or 28S rRNAs. We suggested that these rRNA-like sequences would
interact with ribosomes. The sequences in the sense orientation might mimic rRNA
and bind to ribosomal proteins, whereas those in the antisense or complementary
orientation might form base pairs with rRNA. Because ribosomes are the cellular
structures that translate mRNAs into protein, we proposed that these interactions
would directly affect translation.
In more recent studies, photochemical cross-linking was used to show that
within intact ribosomes various segments of the 18S rRNA are accessible and can
form base pairs with complementary mRNA sequences. In addition, cell-free translation
and transfection studies indicated that these interactions directly affect the
translation of the mRNAs encoding ribosomal protein S15 and the homeodomain protein
Gtx. These mRNAs contain segments complementary to different regions of the 18S
rRNA. Samples of mRNAs prepared from S15-luciferase or Gtx-luciferase fusion
constructs with various degrees of complementarity to the 18S rRNA were translated
in cell-free lysates. For both mRNAs, we found a strong correlation between the
degree of complementarity to the 18S rRNA and inhibition of luciferase activity.
As the degree of complementarity to the 18S rRNA increased, translation of the
S15-luciferase mRNA decreased 2-fold; translation of the Gtx-luciferase mRNA
In addition to examining complementary sequence matches, we are determining
whether rRNA-like sequences in the sense orientation allow some mRNAs to bind
ribosomal proteins and affect translation. We are generating reagents for this
analysis, including ribosomal proteins expressed as recombinant proteins and
antibodies to individual ribosomal proteins.
Our studies provide some of the first examples indicating that the translation
of some eukaryotic mRNAs is directly controlled by base pairing to the 18S rRNA.
One of our goals is to determine the actual extent to which base pairing to rRNA
affects the overall pattern of gene expression.
Atkins, A.R., Osborne, M.J., Lashuel, H.A., Edelman, G.M., Wright, P.E.,
Cunningham, B.A., Dyson, H.J. Association between the first two immunoglobulin-like
domains of the neural cell adhesion molecule N-CAM. FEBS Lett. 451:162, 1999.
Hu, M.C.-Y., Tranque, P., Edelman, G.M., Mauro, V.P. rRNA-complementarity
in the 5´ untranslated region of mRNA specifying the Gtx homeodomain protein:
Evidence that base-pairing to 18S rRNA affects translational efficiency. Proc.
Natl. Acad. Sci. U. S. A. 96:1339, 1999.
Krushel, L.A., Cunningham, B.A., Edelman, G.M., Crossin, K.L. NF-κB
activity is induced by neural cell adhesion molecule binding to neurons and astrocytes.
J. Biol. Chem. 274:2432, 1999.
Little, E.B., Edelman, G.M., Cunningham, B.A. Palmitoylation of the
cytoplasmic domain of the neural cell adhesion molecule N-CAM serves as an anchor
to cellular membranes. Cell Adhes. Commun. 6:415, 1998.
Meech, R., Kallunki, P., Edelman, G.M., Jones, F.S. A binding site
for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene
expression by Pax-6 and bone morphogenetic proteins. Proc. Natl. Acad. Sci. U.
S. A. 96:2420, 1999.
Tranque, P., Hu, M.C.-Y., Edelman, G.M., Mauro, V.P. rRNA complementarity
within mRNAs: A possible basis for mRNA-ribosome interactions and translational
control. Proc. Natl. Acad. Sci. U. S. A. 95:12238, 1998.