trans

262 INTRACELLULAR SIGNALING
and induces differentiation, a process that is
accompanied by a 2–3-fold increase in the
level of intracellular cAMP. Addition of membrane
permeable cAMP analogs or phosphodiesterase
inhibitors to parasite cultures also
mimics the effects of SIF, suggesting that differentiation
is mediated by the cAMP signaling
pathway.
In T. cruzi cAMP also has a role in differentiation.
Addition of cAMP analogs to epimastigote
cultures promotes metacyclogenesis.
In addition, two putative activating ligands
that result in both increased cAMP levels and
enhanced differentiation have been identified.
One is the globin-derived factor (GDF), a small
peptide which arises from proteolytic cleavage
of D -globin within the hindgut of the
insect vector. Administration of this peptide
in vitro results in activation of parasite AC in
membrane fractions and enhanced differentiation
from epimastigotes to metacyclic trypomastigotes.
When infected triatomine bugs
are fed on plasma rather than blood, parasite
differentiation is inhibited unless the GDF
peptide is included. In the same way, metacyclic
trypomastigotes proteolytically degrade
fibronectin into several peptides, two of which
appear to activate AC and initiate transition to
the amastigote stage.
Although putative activators have now been
identified, more detailed dissection of this
signal transduction pathway has not been
reported. In addition, many of the experiments
used to investigate the role of the AC pathway
in T. cruzi were designed on the assumption
that the parasite enzyme would conform
to the structure of mammalian G-proteindependent
ACs, and respond similarly to activators
and inhibitors. As outlined above, so far
this has not proved to be the case. For example,
the T. cruzi enzyme lacks the sequences
necessary for G and G binding, and in biochemical
assays does not respond to forskolin.
The interpretations drawn from these earlier
reports should now be treated with caution, and
a re-evaluation of the molecular basis of cAMPmediated
signaling in T. cruzi is warranted.
Guanylyl cyclase activity in
Plasmodium
In the human malaria parasite P. falciparum,
GC activity is associated with two large
proteins (PfGC and PfGC) that have topological
features reminiscent of mammalian
membrane-localized ACs (Figure 11.5). Unusually,
these proteins both appear to be bifunctional
in that their amino-terminal domains
have strong similarities to P-type ATPases,
including a region predicted to contain ten
membrane-spanning helices. The sequence
and structure of the carboxy terminal regions
of these proteins conforms to those expected
of a mammalian AC, with two sets of six transmembrane
sequences, each followed by the
C1 and C2 catalytic domains. However, the
amino acids that are thought to be enzymatically
important, and are normally present in
the C2 domain of mammalian AC, are located
in the C1 domain of the P. falciparum proteins.
Likewise, amino acids that are present in the
C1 domain of the mammalian enzyme, and
that have a mechanistic role, are located in the
C2 domain of the malarial cyclases. In addition,
the key residues in the P. falciparum enzyme
that determine purine specificity are characteristic
of GC.
Biochemical analysis of the C1 and C2
domains has confirmed that they have GC,
but not AC, activity. In addition, some of the
residues in the cation-binding helices of the
ATPase domain, including an aspartate essential
for ion transport, are not present. As a consequence,
it has been postulated that these
proteins may associate with organic molecules
rather than inorganic ions. Therefore the ATPase
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA