Membrane and Secretory Proteins 
circulation by specific t-PA receptors on liver 
cells. 
The t-PA polypeptide is composed of a number 
of independent functional domains, which in- 
clude a hydrophobic signal sequence, a short pro 
segment, a finger domain having homology to the 
fibrin-binding finger domains of fibronectin, an 
epidermal growth factor (EGF)-like domain, two 
kringle structures, and finally the catalytic do- 
main that is homologous to other members of the 
serine protease family. We have shown that the 
finger and/or EGF-like domains are involved in 
the initial high-affinity binding of t-PA to fibrin, 
while stimulation of t-PA activity involves second- 
ary, lower affinity interactions of fibrin with ei- 
ther of the two kringle domains. The binding of 
t-PA to specific receptors on hepatic cells also 
involves sequences within the finger and/or EGF- 
like domains. 
Although the three-dimensional structure of 
t-PA has not been elucidated, we have been able 
to model the finger domain, the EGF-like domain, 
the kringle domains, and the light-chain/inhibi- 
tor complex using the known structures of homol- 
ogous proteins. Site-directed mutants designed 
using these proposed structures have provided 
information about the individual amino acid se- 
quences that interact with the effector mole- 
cules. For example, we have generated variant 
enzymes that are efficient, fibrin-stimulated plas- 
minogen activators but are resistant to inhibition 
by a variety of serpins (including PAI- 1 ) or do not 
bind to the t-PA receptor(s) involved in clearance 
of the enzyme in the liver. Because these mutant 
enzymes should have an extended effective life in 
the circulation, they may have significant poten- 
tial for use in thrombolytic therapy of patients 
with myocardial infarction. 
Protein Folding Within Cells 
Until recently it was widely assumed that the 
folding and assembly of newly translocated poly- 
peptides into their tertiary and quaternary struc- 
tures is a spontaneous process that does not in- 
volve the intervention of other cellular proteins. 
It is now apparent, however, that members of 
heat-shock protein families, including the ER- 
resident protein BiP, are intimately involved in 
facilitating the folding and assembly of nascent 
polypeptides. 
BiP binds transiently to a variety of wild-type 
membrane and secretory proteins and more per- 
manently to malfolded proteins that are trapped 
in the ER. These observations led to the hypothe- 
sis that BiP plays a role in folding in the ER lumen. 
Analysis of the interaction of BiP with HA mutants 
that lack various structural domains has demon- 
strated that BiP binds to amino acids that form the 
trimeric stalk domain in the folded HA molecule. 
BiP also appears to bind to and stabilize partially 
folded polypeptides in a state competent for fur- 
ther folding and oligomeric assembly. 
Cloning and sequencing of cDNAs encoding 
BiP from mammalian and yeast cells have re- 
vealed an extremely high degree of evolutionary 
conservation. The yeast BiP gene is essential for 
cell viability and, like its mammalian counter- 
part, can be induced by the accumulation of un- 
folded proteins in the ER. The yeast gene differs 
from the mammalian gene, however, in also re- 
sponding to heat shock. All the information re- 
quired for accurate transcriptional regulation of 
yeast BiP is contained within a 230-base pair re- 
gion directly upstream of the initiation codon. 
This sequence contains a functional consensus 
heat-shock element and an element that is respon- 
sible for induction of yeast BiP mRNA following 
accumulation of unfolded proteins in the ER. 
This unfolded protein response (UPR) element 
displays homology to conserved sequences pres- 
ent in the promoters of mammalian glucose-regu- 
lated proteins. 
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