Articles 
Chan, S.J., Nagamatsu, S., Cao, Q.-P., and Steiner, 
D.F. 1992. Structure and evolution of insulin and 
insulin-like growth factors in chordates. Prog 
Brain Res 92:15-24. 
Chan, S.J., Oliva, A.A., Jr., LaMendola, J., Grens, 
A., Bode, H., and Steiner, D.F. 1992. Conserva- 
tion of the prohormone convertase gene family in 
metazoa: analysis of cDNAs encoding a PC3-like 
protein from hydra. Proc Natl Acad Set USA 
89:6678-6682. 
Nagamatsu, S., Nishi, M., and Steiner, D.F. 1991. 
Biosynthesis of islet amyloid polypeptide. Ele- 
vated expression in mouse |STC3 cells. / Biol 
Chem 266A^lil-\ilA\. 
Nagamatsu, S., and Steiner, D.F. 1992. Altered glu- 
cose regulation of insulin biosynthesis in insulin- 
oma cells: mouse |STC3 cells secrete insulin- 
related peptides predominantly via a constitutive 
pathway. Endocrinology 130:748-754. 
Nishi, M., Sanke, T., Ohagi, S., Ekawa, K., Wakasaki, 
H., Nanjo, K., Bell, G.I., and Steiner, D.F. 1992. 
Molecular biology of islet amyloid polypeptide. 
Diabetes Res Clin Pract 15:37-44. 
Ohagi, S., LaMendola, J., LeBeau, M.M., Espinosa, 
R., Ill, Takeda, J., Smeekens, S.P., Chan, S.J., 
and Steiner, D.F. 1992. Identification and analy- 
sis of the gene encoding human PC2, a prohor- 
mone convertase expressed in neuroendocrine 
tissues. Proc Natl Acad Sci USA 89:4977-4981 . 
Ohagi, S., Nishi, M., Bell, G.I., Ensinck, J.W., and 
Steiner, D.F. 1991. Sequences of islet amyloid 
polypeptide precursors of an Old World monkey, 
the pig-tailed macaque (Macaca nemistrine), 
and the dog (Canis familiaris). Diabetologia 
34:555-558. 
Qian, P., Frankfater, A., Steiner, D.F., Bajkowski, 
A.S., and Chan, S.J. 1991. Characterization of 
multiple cathepsin B mRNAs in murine B 1 6a mela- 
noma. Anticancer Res 11:1445-1452. 
Shennan, K.I.J., Seal, A.J., Smeekens, S.P., Steiner, 
D.F., and Docherty, K. 1991. Site-directed muta- 
genesis and expression of PC2 in microinjected 
Xenopus oocytes. / Biol Chem 266:24011- 
24017. 
Smeekens, S.P., Chan, S.J., and Steiner, D.F. 
1992. The biosynthesis and processing of neuro- 
endocrine peptides: identification of proprotein 
convertases involved in intravesicular processing. 
Prog Brain Res 92:235-246. 
Steiner, D.F. 1991- Prohormone convertases re- 
vealed at last. Curr Biol 1:375-377. 
MEMBRANE TRANSPORT PROCESSES 
Michael J. Welsh, M.D., Investigator 
Cystic fibrosis (CP) is a common lethal genetic 
disease of Caucasians that is characterized by abnor- 
mal electrolyte transport in several organs, includ- 
ing the lung. CF is caused by mutations in the gene 
that encodes the cystic fibrosis transmembrane con- 
ductance regulator (CFTR). Amino acid sequence 
analysis and comparison with other proteins sug- 
gested that CFTR contains five domains: two mem- 
brane-spanning domains; one R domain, which con- 
tains several consensus phosphorylation sequences; 
and two nucleotide-binding domains, which are 
predicted to interact with ATP. Previous work from 
this and other laboratories indicated that CFTR is a 
regulated chloride channel. 
To understand the biology of CFTR and the patho- 
genesis of CF, Dr. Welsh's laboratory did several 
studies focused on the function of CFTR and its indi- 
vidual domains. Previous studies had shown that the 
CFTR chloride channel is regulated by agents that 
increase cellular cAMP. In addition, a CFTR mutant 
in which the R domain was deleted produced a 
channel that was open, even without an increase in 
cellular levels of cAMP. To understand further how 
the R domain regulates CFTR, the laboratory studied 
excised, inside-out patches of membrane from cells 
expressing recombinant CFTR. Addition of the cata- 
lytic subunit of cAMP-dependent protein kinase 
(PKA) and ATP to the cytosolic surface activated 
CFTR chloride channels. This indicated that the 
channel is regulated by phosphorylation. 
To identify the R domain as the site of phosphory- 
lation and to identify specific residues responsible 
for channel activation, each of the 10 potential PKA 
phosphorylation sites was mutated individually. 
The consensus sequence for PKA-dependent phos- 
phorylation is A:g/Lys-Arg/Lys-X-Ser/Thr; at each 
potential site, Ser/Thr was mutated to Ala. The stud- 
ies showed that upon stimulation with cAMP, four 
serines were phosphorylated in vivo; all are located 
in the R domain. Mutation of any single serine did 
CELL BIOLOGY AND REGULATION 
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