Evidence that the C Terminus of the A Subunit Suppresses Thyrotropin Receptor Constitutive Activity

Abstract

The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261-418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.

The Thyroid Disruptor 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl)-Ethane Appears to Be an Uncompetitive Inverse Agonist for the Thyrotropin Receptor

“…Trypsin is expected to increase the TSHr constitutive activity by removing the inhibitory effect of the ECD. In line with data by Van Sande et al (1996) and Chen et al (2003), trypsin increased significantly the TSHr constitutive activity, but it did not reduce DDT and Aroclor 1254 inhibitory effects (Fig. 3).…”

“…That such an effect may really occur was actually demonstrated by Zhang et al (2000), who found that activation of the TSHr is secondary to "beheading" in N-terminal truncated mutants. Recently, Chen et al (2003) have narrowed down the inhibitory effect of the ectodomain to its C terminus, indicating a cluster of lysine (Lys287, Lys290, Lys291) and arginine (Arg293) residues as a possible target for the functional effect of trypsin.…”

Significance of Ectodomain Cysteine Boxes 2 and 3 for the Activation Mechanism of the Thyroid-stimulating Hormone Receptor

“…In support of this hypothesis, Chen et al (27) have shown that a cluster of positively charged hydrophilic amino acids in C-b2 forms the site for tryptic clipping, which also results in partial receptor activation. Such an activation mechanism requires flanking anchor points that constrain the basal WT TSHR conformation.…”

“…Based on the model of a tightly packed structural arrangement of C-b2 and C-b3 via disulfide bridges (26) and considering activation by tryptic clipping (27), we hypothesized that apart from Ser-281 additional amino acids in C-b2 could also be sensitive for constitutive activation by mutations. Therefore, we tested by single alanine substitution whether further hydrophilic residues of C-b2, Lys-287, Asn-288, Gln-289, Lys-290, Lys-291, and Arg-293, are involved in constraining the WT TSHR conformation.…”

“…Indeed, mutations at Pro-400 and Pro-407 also lead to constitutive activation of the TSHR. Moreover, tryptic clipping within C-b2 at an undefined site in the region of a cluster of positively charged amino acids (Lys-287-Arg-293) close to Ser-281 also leads to receptor activation (27). Considering the tightly packed structural arrangement of C-b2 and C-b3 via disulfide bridges, we assumed that mutation of additional amino acids within C-b2 might also lead to constitutive activation.…”