Mouse ADAM33: two splice variants differ in protein maturation and localization.
We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 (alpha) and 906 (beta), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor-like domains. Proteins of approximately 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the approximately 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, approximately 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature approximately 100-kD, a minority of the approximately 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H(f). The approximately 110-kD form was endoglycosidase H(f)-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded beta splice variant are unlikely to occur in mouse lung.
Umland
Shelby P
SP
Departments of Allergy and of Statistics, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. shelby.umland@spcorp.com
Wan
Yuntao
Y
Shah
Himanshu
H
Garlisi
Charles G
CG
Devito
Kristine E
KE
Braunschweiger
Karen
K
Gheyas
Ferdous
F
Del Mastro
Richard
R
D051722:ADAM Proteins; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D019483:Disintegrins / Q000235:genetics / Q000378:metabolism; D005091:Exons; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000502:physiology*; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D019483:Disintegrins; D000001:Calcimycin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D051722:ADAM Proteins; C450441:ADAM33 protein, human; C499183:Adam33 protein, mouse; D008666:Metalloendopeptidases