BIOSYNTHESIS AND ACTIONS OF ISLET HORMONES 
Donald F. Steiner, M.D., Senior Investigator 
Progress during the past year includes studies on 
1) basic cellular mechanisms of insulin production 
and action, 2) islet cell secretory products and their 
precursors, 3) mutations in insulin and insulin re- 
ceptor genes in humans with diabetes or other met- 
abolic syndromes, and 4) the structure and func- 
tion of the insulin receptor. 
I. Biosynthesis of Islet Hormones. 
A. Prohormone-converting enzymes. Early studies 
from Dr. Steiner's laboratory, as well as others, have 
established that prohormone processing usually 
occurs via cleavage at pairs of basic residues, such 
as Lys-Arg or Arg-Arg, by a trypsin-like endoprotease. 
A carboxypeptidase B-like exopeptidase then excises 
the basic residues from the newly created carboxyl 
termini. Processing occurs mainly in secretory 
vesicles and also includes carboxyl-terminal ami- 
dation of some peptides. The nature of the mam- 
malian processing endoprotease(s) has been con- 
troversial. A number of candidate enzymes have 
been proposed, but few, if any, of these have been 
shown to participate in vivo, except for the Kex2 
enzyme of yeast, which cleaves the a-factor pre- 
cursor. 
Several years ago a project was begun to identify 
cDNAs from animal tissues that would complement 
the KEX2 mutation in yeast. Direct screening of 
islet libraries yielded no positives. Vectors were 
then constructed for expressing islet cDNA libraries 
in yeast, and suitable Kex2-minus strains of yeast 
were developed. These efforts also failed to pro- 
duce any positives. A new approach was then 
explored that involved the use of polymerase 
chain reaction (PGR) to search for sequences re- 
lated to those of Kex2 in insulinoma mRNA. This 
approach has proven successful, and a cDNA that 
appears to be related to Kex2 from human in- 
sulinoma mRNA has recently been identified. The 
encoded protein retains the necessary catalytic resi- 
dues for proteolysis, suggesting that it is an enzyme 
related to Kex2 and/or subtilisin. This full-length 
cDNA is currently being expressed in various ani- 
mal cells, as well as in yeast and Xenopus oocytes, 
to explore the nature of the proteolytic activity that 
it may encode. A membrane-bound form of car- 
boxypeptidase B has also recently been cloned in 
collaboration with Dr. Randal Skidgel (University of 
Illinois). 
B. Sorting mechanisms of beta cells. Several years 
ago studies were initiated on a mutant proinsulin 
gene associated with familial hyperproinsulinemia. 
A single Asp for His substitution at position 10 of 
the B chain was found. Synthesis of the corre- 
sponding mutant insulin molecule by Dr. Panayotis 
Katsoyannis and co-workers revealed a binding and 
biological potency four- to fivefold greater than that 
of normal human insulin. A series of biosynthetic 
studies was then begun on islets from transgenic 
mice harboring this mutant human gene [in collab- 
oration with Dr. Robert E. Hammer (HHMI, Univer- 
sity of Texas Southwestern Medical Center at Dal- 
las)]. The secretion of unprocessed mutant 
proinsulin into the medium is about five- to sixfold 
greater than that of the normal mouse pro- 
hormones. A significant fraction of the mutant 
human proinsulin appears to enter an unregulated 
or constitutive pathway. Further studies are in prog- 
ress to assess the possible role of the elevated re- 
ceptor-binding affinity of the mutant proinsulin in 
its aberrant sorting behavior. 
II. Islet Amyloid Polypeptide (LVPP). 
Recent studies have shown that amyloid deposits 
present in the islets of type II diabetics consist of a 
neuropeptide structurally related to calcitonin 
gene-related peptide (CGRP). Several laboratories 
reported amino acid sequences for this 37-amino 
acid peptide (also called amylin) from diabetic 
human as well as cat pancreas. Studies by Dr. Per 
Westermark and his associates in Uppsala have 
shown that this peptide is present in the beta cells 
and is stored in the insulin granules. To gain a bet- 
ter understanding of its biosynthesis. Dr. Steiner 
and his colleagues isolated a full-length cDNA en- 
coding the LAPP precursor from a human in- 
sulinoma library. The predicted amino acid se- 
quence revealed a polypeptide of 89 amino acids, 
beginning with a typical signal peptide followed by 
a prohormone containing the lAPP moiety brack- 
eted at each end by pairs of basic residues. A gly- 
cine residue just beyond the carboxyl terminus of 
LAPP indicated that this peptide would likely be 
amidated during its processing in the beta cell. Fur- 
ther comparative studies in cats, rats, mice, and 
guinea pigs have shown that the LAPP sequence is 
highly conserved, while the remainder of the pre- 
cursor shows a much higher level of variation, sug- 
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