Insulin and the Islets of Langerhans 
processing enzyme. PC2 is more abundant in islet 
(S-cells, while PC 3 predominates in the anterior 
pituitary. Expression of PC2 in Xenopus oocytes 
gives rise to a 68-kDa protein that is active on 
substrates having characteristic sequences for 
prohormone cleavage recognition (i.e., Lys-Arg 
or Arg-Arg) . The proteolytic activity requires cal- 
cium ions and is highest at pH 5.5. Other expres- 
sion studies carried out in collaboration with 
Gary Thomas and his co-workers at the Vollum 
Institute indicate that PC 2 and PC 3 separately or 
together can appropriately process both proopio- 
melanocortin or proinsulin. Studies are now in 
progress on the genes encoding these proteases 
and their evolutionary origins, as well as on the 
synthesis, sorting, and activation of PC2 and/or 
PC3 in various neuroendocrine cells. 
Regulation of Insulin Secretion 
We have recently isolated and characterized 
cDNA and genomic clones encoding several volt- 
age-dependent K"^ channel isoforms expressed in 
human islets or insulin-producing tumors. These 
channels are related to the Shaker family of Dro- 
sophila K"^ channels. Their electrophysiological 
characteristics have been examined by voltage 
clamp recordings of oocytes injected with syn- 
thetic mRNA. Efforts are also under way to iden- 
tify and characterize an ATP-dependent potas- 
sium channel believed to play a key role in 
initiating /S-cell depolarization in response to 
glucose or sulfonylureas. 
Insulin Receptor Studies 
Studies nearing completion have revealed an 
interesting and potentially important aspect of 
insulin proreceptor processing, namely that un- 
cleaved proreceptors having exon 1 1 (which en- 
codes 1 2 amino acids near the carboxyl terminus 
of the a-subunit) are fully functional, while those 
lacking this exon (due to alternative splicing of 
the mRNA) are markedly reduced in their afi&nity 
for insulin. Since most cells express receptors 
without exon 1 1 , failure to cleave the prorecep- 
tor (as occurs in a family we have studied that has 
mutant proreceptors that cannot be processed) 
leads to severe insulin-resistant diabetes. The 
liver, kidney, islet ;8-cells, and placenta are the 
only tissues that express insulin receptors with 
exon 1 1 , suggesting that alternative splicing may 
be physiologically relevant. 
Biosynthesis of Islet Amyloid 
Polypeptide (lAPP) 
LAPP, or amylin, a recently discovered product 
of the (8-cell, is a peptide related to CGRP (calci- 
tonin gene-related peptide) that is found in amy- 
loid deposits in the islets of elderly diabetics and 
may play a role in impairing |8-cell function. We 
have characterized the gene and cDNAs encoding 
the precursor of lAPP in humans and several other 
mammalian species. We are currently studying a 
transgenic mouse model to identify factors that 
may contribute to amyloid deposition (in collabo- 
ration with Niles Fox of the Lilly Research 
Laboratories) . 
Evolution of Insulin and Insulin-like 
Growth Factors 
We have used the polymerase chain reaction to 
identify genes encoding insulin or the closely re- 
lated insulin-like growth factors IGF-I and -II in 
primitive vertebrates. The identification of a hy- 
brid insulin/IGF molecule in amphioxus, a pro- 
tochordate, suggests that the insulin-like growth 
factors diverged from an ancestral preproinsulin- 
like protein in the very earliest stages of verte- 
brate evolution (about 600 million years ago). 
Studies nearing completion on the identification 
and structure of insulin and IGF receptors in 
these lower forms also lend support to this model 
for the origin of the insulin-like growth factors as 
unique vertebrate growth regulators. 
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