MOLECULAR NEUROIMMUNOLOGY 
Donald G. Payan, M.D., Assistant Investigator 
Dr. Payan is examining the biochemical activities 
of neuropeptides and neural mediators active in 
various phases of inflammation. The principal on- 
going experimental effort is to analyze the prolifera- 
tion and growth-promoting properties of these fac- 
tors, through a detailed study of their receptors and 
associated signaling pathways. To this end, the re- 
ceptor for the neuropeptide substance P (SP) and 
the signaling pathways associated with the hista- 
mine receptor are being actively investigated. The 
principal achievements of Dr. Payan and his col- 
leagues in the past year include 1) a detailed analy- 
sis, using antipeptide antibodies, of the epitopes on 
the SP receptor that may be coupled to peptide- 
induced signaling; 2) the solubilization and charac- 
terization of the histamine (H^) receptor from cul- 
tured smooth muscle cells; and 3) the study of the 
multiple signaling pathways of receptors. 
I. Tachykinin Receptor Cross Talk. 
Dr. Payan and his colleagues chemically synthe- 
sized peptides that correspond to the four extracel- 
lular domains of the predicted bovine substance K 
(SK) receptor protein and raised specific polyclonal 
antibodies against these peptides. These antibodies 
were then tested for both functional and structural 
recognition of epitopes on the SP receptor on rat 
AR42J pancreatic cells, which express only SP, and 
not SK, receptors. Antibodies directed against the 
second and fourth external domains of the pre- 
dicted SK receptor specifically recognized a 58 kDa 
protein on AR42J cells, of similar molecular weight 
to that of Bolton-Hunter ^^^i.iabeled SP ([^^^ijbh. 
SP)-crosslinked membrane proteins of AR42J cells 
and the previously reported SP receptor on human 
IM9 lymphoblasts. These antibodies also inhibited 
specific [^^^I]BH-SP binding on both AR42J and IM9 
cells. Furthermore, these antibodies also inhibited 
SP-induced mobilization of intracellular Ca^"*" 
([Ca^^].) on AR42J cells. These data suggest homol- 
ogies between the extracellular structures of the SP 
and SK receptors and imply that the second and 
fourth domains of these receptors may share 
ligand-binding domains. These data also suggest 
that the cross talk between SP- and SK-mediated re- 
sponses may be explained by the homology of the 
extracellular structures between both receptor pro- 
teins, as well as the homologies of their peptide 
carboxyl termini. 
These structural similarities between the SP and 
SK receptors and the fact that both receptors trans- 
duce their signals through similar G protein- 
coupled mechanisms are being used to isolate the 
gene for the SP receptor, using the polymerase 
chain reaction method and low-stringency hybrid- 
ization techniques. 
IL Solubilization and Characterization of Histamine 
Receptors. 
Dr. Payan and Dr. M. Mitsuhashi previously 
showed that the cultured smooth muscle cell line 
DDTjMF-2 expresses a large number (9.7 x 10*' re- 
ceptors/cell) of functional H^^-type receptors. The 
most recent work, using two diJBFerent binding as- 
says (gel filtration and polyethylene glycol precipi- 
tation), indicated that the [^H]pyrilamine-binding 
activity was solubilized by 1% digitonin, with bind- 
ing characteristics similar to those of intact cells. 
The solubilized proteins were then purified by se- 
quential gel filtration, chromatofocusing, and re- 
verse-phase high-pressure liquid chromatography. 
The calculated molecular weight of this purified 
pyrilamine-binding protein was 38-40 kDa on so- 
dium dodecyl sulfate-polyacrylamide gel electro- 
phoresis. The binding of [^H]pyrilamine to these 
38-40 kDa proteins indicated a single class of bind- 
ing site, with a of 288 nM, which is equivalent to 
that of intact cells and digitonin-solubilized pro- 
teins. The computer analysis Scatfit also indicated 
that one molecule of [^H]pyrilamine bound to one 
molecule of purified protein. Furthermore, a poly- 
clonal antibody raised against the purified protein 
recognized the 38-40 kDa band by Western blot- 
ting techniques, specifically bound to the cell 
surface of DDTjMF-2 cells, and inhibited [^Hjpyrila- 
mine binding to these cells in a dose-dependent man- 
ner. These data strongly suggest that the purified 38- 
40 kDa protein is part of an antagonist-binding 
domain on the receptor on DDT^MF-2 cells. 
in. Multiple Signaling Pathways After Histamine 
Binding. 
To analyze the complex activities of H^-receptor 
activation on neutrophils, human HL-60 promyelo- 
cyte leukemia cells were differentiated by Drs. 
Payan and Mitsuhashi into neutrophils by incuba- 
tion with dimethylsulfoxide, loaded with the Ca^^- 
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