Aggregation of human wild-type and H27A-prolactin in cells and in solution: roles of Zn(2+), Cu(2+), and pH.
Aggregation of hormones is an important step in the formation of secretory granules that results in concentration of hormones. In transfected AtT20 cells, but not COS cells, Lubrol-insoluble aggregates of human prolactin (PRL) accumulated within 30 min after synthesis. Aggregation in AtT20 cells was reduced by incubation with 30 microM chloroquine, which neutralizes intracellular compartments, and was slowed by incubation with diethyldithiocarbamate, which chelates Cu(2+) and Zn(2+). H27A-PRL aggregated in AtT20 cells as well as wild-type PRL, indicating that a high affinity Zn(2+)-binding site is not necessary. In solution, purified recombinant human PRL was precipitated by 20 microM Cu(2+) or Zn(2+). In solution without polyethylene glycol there was no precipitation with acidic pH alone, precipitation with Zn(2+) was most effective at neutral pH, and the ratio of Zn(2+) to PRL was greater than 1 in the precipitate. In solution with polyethylene glycol, precipitation occurred with acidic pH, precipitation with Zn(2+) occurred effectively at acidic pH, and the ratio of Zn(2+) to PRL was less than 1. The aggregates obtained in polyethylene glycol are therefore better models for aggregates in cells. Unlike human PRL, aggregation of rat PRL has been shown to occur at neutral pH in cells and in solution, and therefore these two similar proteins form aggregates that are the cores of secretory granules in ways that are not completely identical.
Sankoorikal
Binu-John
BJ
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Zhu
Yong Lian
YL
Hodsdon
Michael E
ME
Lolis
Elias
E
Dannies
Priscilla S
PS
D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011388:Prolactin / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins; D012995:Solubility; D012996:Solutions; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000737:chemistry / Q000502:physiology*
D002614:Chelating Agents; D011994:Recombinant Proteins; D012996:Solutions; D011092:Polyethylene Glycols; D003300:Copper; D011388:Prolactin; D015032:Zinc; D002118:Calcium