trans

260 INTRACELLULAR SIGNALING
ESAG4 (expression site associated gene 4),
are localized close to the ends of chromosomes
in the telomeric variant surface glycoprotein
(VSG) expression sites (for review, see
Chapter 5). These genes are expressed specifically
in the mammalian bloodstream stage
of the life cycle. The second group of genes,
termed GRESAG4s (genes related to ESAG4),
are widely dispersed in the genome and can
occur in clusters, or in single copies. These
genes appear to be constitutively expressed. In
T. cruzi, sequencing data indicate the presence
of a polymorphic family of more than 30 AC
genes. These genes are localized on at least six
chromosomes and are scattered rather than
clustered. AC pseudogenes have also been
identified in T. cruzi.
It has been suggested that trypanosomatid
ACs are activated directly by external ligands,
with the extracellular domain acting as the
receptor, similar to mammalian membranebound
GCs (Figure 11.4A, B). This direct activation
would negate the requirement for
G-protein-dependent activation. Consistent
with this, heterotrimeric G-protein subunit
homologs have not yet been identified by
any of the three trypanosomatid Genome
Projects, and characteristic G-protein-binding
sequences are absent from the trypanosomatid
ACs. In contrast to the conserved catalytic
domains of trypanosome ACs, the extracellular
regions are extremely divergent at the
sequence level. This heterogeneity may play
a role in immune evasion, or may reflect functional
differences between isoforms, such as
ligand-binding specificity. It is also possible
that both of these forces have acted during
evolution to generate the diversity of the AC
repertoire within the constraints necessary for
maintaining receptor function.
Trypanosome ACs are inactive as monomers,
and activity is dependent on dimer formation.
With members of the T. brucei GRESAG4
family, recombinant forms of the catalytic
domains can dimerize spontaneously; however
this does not increase activity above basal
levels. Activity is considerably enhanced,
though, when a yeast leucine zipper sequence
is added to the amino terminus of these
catalytic domains. The leucine zipper may
promote a dimer conformation that is more
favorable for activity, perhaps mimicking
the effects of ligand binding to the extracellular
domain of the native protein. Since the
catalytic domain is formed from two homologous
polypeptides, the trypanosomal enzyme
must have two symmetrical active sites.
At each active site, binding of the ATPcomplexed
metal ion is contributed by one
chain of the homodimer, while purine-binding,
Mg 2 -binding and catalytic residues are contributed
by the other. In this type of enzyme,
the second active site corresponds to the
forskolin-binding site of the mammalian
heterodimeric AC. This model has been confirmed
following the determination of the
structure of a T. brucei AC catalytic domain
by X-ray crystallography and by further biochemical
and mutagenesis analyses. In terms
of structure, the catalytic domain of the
T. brucei AC closely resembles that of the
mammalian enzyme, although it does contain
an additional insertion sequence that forms
a unique motif called the -subdomain. The
sequence of the -subdomain is very highly
conserved among trypanosomatid ACs. It is
located in a position that corresponds to a site
where regulatory cofactors interact with the
mammalian AC, and with membrane-bound
GCs. Most interestingly, the observation that
D-DTT can bind in a stereospecific manner
to a pocket below the -subdomain has led
to the tentative suggestion that in vivo this
region may act as an allosteric control site,
specific for other small molecules that have a
regulatory role.
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA