PROBING THE BIMOLECULAR INTERACTIONS OF PARATHYROID-HORMONE AND THE HUMAN PARATHYROID-HORMONE PARATHYROID HORMONE-RELATED PROTEIN-RECEPTOR .2. CLONING, CHARACTERIZATION, AND PHOTOAFFINITY-LABELING OF THE RECOMBINANT HUMAN RECEPTOR

被引：39

作者：

ADAMS, AE

PINES, M

NAKAMOTO, C

BEHAR, V

YANG, QM

BESSALLE, R

CHOREV, M

ROSENBLATT, M

LEVINE, MA

SUVA, LJ

机构：

[1] BETH ISRAEL HOSP,DEPT MED,HARVARD THORNDIKE & CHARLES A DANA RES LABS,DIV BONE & MINERAL METAB,BOSTON,MA 02215

[2] HARVARD UNIV,SCH MED,BOSTON,MA 02215

[3] HARVARD UNIV,SCH MED,MIT,DIV HLTH SCI & TECHNOL,BOSTON,MA 02215

[4] JOHNS HOPKINS UNIV,SCH MED,DEPT MED,DIV ENDOCRINOL & METAB,BALTIMORE,MD 21205

来源：

BIOCHEMISTRY | 1995年 / 34卷 / 33期

关键词：

D O I：

10.1021/bi00033a030

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and crosslinking of a benzophenone-substituted PTH analog, [Nle(8,18),Lys(13)(epsilon-pBz(2)),L-2-Nal(23),Tyr(34)]bPTH(1-34)-NH2 (K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of similar to 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of I-125-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Re, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.

引用

页码：10553 / 10559

页数：7

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