Post-translational Processing and Renal Expression of Mouse Indian Hedgehog

doi:10.1074/jbc.272.13.8466

Post-translational Processing and Renal Expression of Mouse Indian Hedgehog*

From the Departments of ^‡ Pediatrics,
^∥ Internal Medicine,
^¶ Pediatric Urology, and
** Pathology, and the
^§ Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676

^‡‡ To whom correspondence should be addressed:
Division of Nephrology, Dept. of Internal Medicine, University of Michigan Medical School, Rm. 1560, Medical Science Research Bldg. II, Ann Arbor, MI 48109-0676
. Tel.: 313-936-4812; Fax: 313-763-0982; E-mail: lbholzman{at}medmail.med.umich.edu

Abstract

The full-length mouse Indian hedgehog (Ihh) cDNA was cloned from an embryonic 17.5-day kidney library and was used to study the post-translational processing of the peptide and temporal and spatial expression of the transcript. Sequence analysis predicted two putative translation initiation sites. Ihh translation was initiated at both initiation sites when expressed in an in vitro transcription/translation system. Expression of an Ihh mutant demonstrated that the internal translation initiation site was sufficient to produce the mature forms of Ihh. Ihh post-translational processing proceeded in a fashion similar to Sonic and Drosophila hedgehog; the unprocessed form underwent signal peptide cleavage as well as internal proteolytic processing to form a 19-kDa amino-terminal peptide and a 26-kDa carboxyl-terminal peptide. This processing required His³¹³ present in a conserved serine protease motif. Ihh transcript was detected by in situ RNA hybridization as early as 10 days postcoitum (dpc) in developing gut, as early as 14.5 dpc in the cartilage primordium, and in the developing urogenital sinus. In semiquantitative reverse transcription-polymerase chain reaction experiments, Indian hedgehog transcript was first detected in the mouse metanephros at 14.5 dpc; transcript abundance increased with gestational age, becoming maximal in adulthood. In adult kidney, Ihh transcript was detected only in the proximal convoluted tubule and proximal straight tubule.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants DK-47566 (to L. B. H.) and individual National Research Service Award DK-09375 (to R. V.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U85610[GenBank].
↵¹ The abbreviations used are:

Ihh

Indian hedgehog

Hh

hedgehog

Shh

Sonic hedgehog

Dhh

Desert hedgehog

dpc

days postcoitum

PTHrP

parathyroid hormone-related protein

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase

kb

kilobase pair(s)

bp

base pairs

RT-PCR

reverse transcription-polymerase chain reaction.
- Received November 4, 1996.