Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor
Gd. Chazenbalk et al., Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor, EUR J BIOCH, 268(8), 2001, pp. 2261-2269
To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage
, we sought to convert the noncleaving luteinizing hormone (LH) receptor (L
HR) into a cleaved, two-subunit molecule. For this purpose, we generated a
series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, li
gand binding receptors on the cell surface was determined by covalent I-125
-labeled hCG crosslinking to intact, stably transfected mammalian cells. We
first targeted a cluster of three N-linked glycans in the LHR (N295, N303,
N317) in a region corresponding to the primary TSHR cleavage site, which h
as only one N-linked glycan. Elimination by mutagenesis of the most strateg
ic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavag
e. Removal of the other N-linked glycans had no additive effect. A much gre
ater degree of cleavage (approximate to 50%) was evident in a chimeric LH-T
SHR in which the juxtamembrane segment of the LHR (domain E; amino acids 31
7-367) was replaced with the corresponding domain of the TSHR (residues 363
-418). Similarly cleaving LHR were created using a much smaller component w
ithin this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or
by substitution of the same three amino-acid residues with AAA (LHR-NET317-
319AAA).
In summary, our data alter current concepts regarding TSHR cleavage by sugg
esting limited (not absent) aminoacid specificity in a region important for
TSHR cleavage (GQE367-369). The data also support the concept of a separat
e and distinct downstream cleavage site 2 in the TSHR.