The Production and Actions of Insulin and Other Islet Hormones 
• structure and function of the human insuHn 
receptor, 
• new hormones of the islets — e.g., the re- 
cently discovered amylin peptide, and 
• evolution of insulin and insulin-like growth 
factojs and their related receptors. 
Identification of Prohormone-converting 
Enzymes 
Using recently developed methods of specific 
DNA amplification, we have identified two 
cDNAs (PC2 and PC3) from neuroendocrine cells 
that encode proteolytic domains similar in struc- 
ture to those of Kex2, a yeast prohormone- 
processing enzyme. We are attempting to learn 
more about the expression and function of these 
proteases in i8-cells and to delineate their proteo- 
lytic properties. Our approach is to express the 
proteases in a variety of cells in vitro, along with 
prohormones such as proinsulin and/or POMC 
(proopiomelanocortin) . 
Studies on Potassium Ion Channels of jS-Cells 
Regulation of insulin secretion involves the co- 
ordinated control of ion channels in the /3-cell 
membrane. We have recently isolated and charac- 
terized cDNA and genomic clones encoding a 
voltage-dependent channel isoform expressed 
in human islets and in a human insulin-producing 
tumor. This channel, designated hPCNl, con- 
sists of 613 amino acids and is related to the 
Shaker family of Drosophila channels. Two 
other human channel isoforms that we have 
isolated, hPCN2 and hPCN3, are homologous to 
hPCNl, with 55 and 65 percent amino acid se- 
quence identity, respectively. 
The electrophysiological characteristics of 
hPCNl have been examined by voltage-clamp re- 
cordings of oocytes injected with synthetic 
hPCNl RNA. These have revealed hPCNl to be of 
the delayed rectifier type. The channel could 
function to restore the resting membrane poten- 
tial of j8-cells after depolarization and thereby 
contribute to the regulation of insulin secretion. 
Efforts are also under way to identify and charac- 
terize the ATP-dependent channel that plays a 
key role in initiating i8-cell depolarization in re- 
sponse to glucose. 
Studies of the Insulin Receptor 
We have studied a mutation in the insulin re- 
ceptor protein that gives rise to severe insulin- 
resistant diabetes. The defect in this patient is" a 
single amino acid replacement that prevents the 
receptor precursor from being processed into 
mature receptor a- and /3-subunits on the cell sur- 
face. Studies are now nearing completion on mu- 
tagenesis of the cleavage site of the human insu- 
lin proreceptor to determine the exact structural 
requirements for its processing by a specialized 
cellular protease to generate functional recep- 
tors. We are also using mutagenesis to evaluate 
more fully the functional consequences of disor- 
dered processing of the proreceptor. 
We have developed methods for expressing 
human insulin receptors at very high levels in in- 
sect or mammalian cells in culture. Either normal 
or modified receptor proteins (generated by mu- 
tating the cDNA sequence) can be produced in 
amounts sufficient for detailed study of their 
structures, insulin-binding domains, and biologi- 
cal signaling properties. In other studies, we are 
analyzing the complexity of protein phosphoty- 
rosine phosphatases — cellular enzymes that re- 
verse insulin receptor activation. 
Amylin, a New Product of the /S-CeU 
We recently identified a new j8-cell precursor 
molecule that, when appropriately processed, 
gives rise to a peptide called islet amyloid poly- 
peptide (LAPP), or amylin. This peptide is found 
in amyloid deposits in the islets of elderly dia- 
betics and may play a role in impairing /3-cell 
function. We have characterized the gene encod- 
ing LAPP and have begun studies on its biosynthe- 
sis and secretion in response to glucose and other 
factors. It is produced at a level about 1 percent 
that of insulin and is cosecreted with insulin. 
Studies on LAPP precursors from nine mammalian 
species support the hypothesis that differences in 
the sequence of LAPP in its central region (resi- 
dues 20-29) in different species influences their 
relative susceptibility to the deposition of amy- 
loid in the islets or in insulin-producing tumors 
(insulinomas) . 
The Evolution of Insulin and Insulin-like 
Growth Factors 
We have succeeded in efforts to identify primi- 
tive insulin superfamily-related genes (encoding 
insulin or the closely related insulin-like growth 
factors IGF-I and -II) in primitive vertebrate or- 
ganisms, including cyclostomes (the Atlantic 
hagfish) and amphioxus. The presence 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 molecule in the very earliest stages of verte- 
brate evolution. We are also studying the origin 
and evolution of insulin and IGF receptors in 
these lower forms. 
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