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Science 254:290-293- 
MOLECULAR AND CELL BIOLOGY OF THE ISLETS OF LANGERHANS 
Donald F. Steiner, M.D., Senior Investigator 
During the past year progress has been made in 
studies on the mechanisms of conversion of prohor- 
mones to their biologically active forms in the islets 
of Langerhans and other neuroendocrine tissues. 
PC2 and PC3, two novel subtilisin-related proteases 
identified last year, have been shown to participate 
in the proteolytic processing of two important 
neuroendocrine precursors: proopiomelanocortin 
(POMC) and proinsulin. The gene encoding PC2 
has now been cloned and sequenced, and aspects of 
its expression are being examined. Studies on the 
evolution of insulin, insulin-related growth factors 
(IGFs) and their receptors, and the converting en- 
zymes PC2, PC3, and furin are under way in both 
primitive vertebrates and invertebrates. Studies also 
are in progress on transgenic mice expressing genes 
encoding human islet amyloid polypeptide (lAPP), 
a second product of the jS cell that gives rise to islet 
amyloid deposits in type II diabetics. The structural 
requirements of the insulin receptor precursor- 
processing site have been examined and the effect 
on ligand binding of cleavage between the a and ^ 
subunits has been proved to depend on the receptor 
isoform. 
Subtilisin-related 
Prohormone-converting Enzymes 
Intracellular processing of neuroendocrine and 
peptide hormone precursors usually occurs via 
cleavage at certain pairs of basic residues, i.e., Lys- 
Arg or Arg-Arg, by an endoprotease that cleaves on 
the carboxyl-terminal side of the pair. Carboxypep- 
tidase E or H, an exopeptidase, then removes the 
basic residues from the newly created carboxyl ter- 
mini. In neuroendocrine cells such processing oc- 
curs mainly in newly formed secretory vesicles of 
the regulated pathway. Two mammalian prohor- 
mone-processing endoprotease (s), PC2 and PC3, 
have recently been identified in Dr. Steiner's labora- 
tory. These are serine proteases having a catalytic 
core that is homologous to that of the Kex2 protease 
of yeast, which cleaves both the a-mating and killer 
factor precursors at basic residue pairs. 
The PC2 and PC3 proteases were identified by the 
polymerase chain reaction by using primers based 
on conserved sequences surrounding essential cata- 
lytic residues of Kex2 and the related bacterial sub- 
tilisins. Their tissue distribution indicated that both 
proteases are expressed selectively in neural and 
neuroendocrine tissues. The full-length cDNAs for 
PC2 and PC3 were then expressed in Xenopus oo- 
cytes to define the nature of the encoded proteolytic 
activity. Both proteases have acidic pH optima and 
depend on calcium ions for activity. Vaccinia virus 
vectors have been used to study the activity of PC2 
and PC3 on several neuroendocrine precursors, in 
collaboration with Gary Thomas and his co-workers 
(VoUum Institute, Portland). These studies indicate 
that PC2 and PC3 differ significantly in their proteo- 
lytic site selectivity; PC3 reproduces the pattern of 
cleavage of POMC characteristic of the anterior pitu- 
itary, while PC2 and PC3 together give a pattern 
typical of the intermediate lobe. These results are 
consistent with the patterns of expression of PC3 
and PC2 within the pituitary gland. Similar studies 
in progress on proinsulin indicate that PC2 and PC3 
each preferentially cleave at only one of the two 
basic residue pairs that must be processed in the 
conversion of proinsulin to insulin. 
The gene for human PC2 has been identified and 
characterized. It is a large gene (>130 kbp in 
length) and contains 12 exons. The promoter has 
been partially characterized, and efforts are under 
way to determine how it is regulated and whether 
defects in the PC2 gene may occur in some forms of 
diabetes. 
CELL BIOLOGY AND REGULATION 
115 
