Cloning, sequencing and expression of human TSH receptor

An increasing number of examples in the literature suggest that the in vivo duration of drug action not only depends on macroscopic pharmacokinetic properties like plasma half-life and the time needed to equilibrate between the plasma and the effect compartments, but is also influenced by long-lasting target binding and rebinding. The present review combines information from different research areas and simulations to explore the nature of these mechanisms and the conditions in which they are most prevalent. Simulations reveal that these latter phenomena become especially influential when there is no longer sufficient free drug around to maintain high levels of receptor occupancy. There is not always a direct link between slow dissociation and long-lasting in vivo target protection, as the rate of free drug elimination from the effect compartment is also a key influencing factor. Local phenomena that hinder the diffusion of free drug molecules away from their target may allow them to consecutively bind to the same target and/or targets nearby (denoted as 'rebinding') even when their concentration in the bulk phase has already dropped to insignificant levels. The micro-anatomic properties of many effect compartments are likely to intensify this phenomenon. By mimicking the complexity of tissues, intact cells offer the opportunity to investigate both mechanisms under the same, physiologically relevant conditions.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1476-5381.2010.00936.x

Long‐lasting target binding and rebinding as mechanisms to prolong in vivo drug action

A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen.

G-protein-coupled receptors (GPCRs) have long been believed to activate G proteins only on the cell surface. However, we have recently shown that, in thyroid cells, the GPCR for the thyroid-stimulating hormone (TSH) can continue stimulating cAMP production after cointernalization with TSH. cAMP signaling by internalized TSH receptors (TSHRs) was persistent, whereas that by cell-surface TSHRs was apparently transient, but the reasons for the transient signaling by cell-surface TSHRs were not investigated. Here, we developed and used fluorescence resonance energy transfer (FRET)-based methods to precisely compare the kinetics of TSH binding and dissociation from cell-surface TSHRs with those of the subsequent termination of cAMP signaling directly in living cells. Our results indicate that both TSH binding to human TSHRs expressed in a human embryonic kidney cell line (HEK 293) and the ensuing cAMP signals are rapidly and fully reversible (t(1/2,off)=2.96±1.04 and 2.70±0.73 min, respectively). The FRET measurement of TSH binding was specific, as shown by the lack of a detectable interaction between TSH and the β(2)-adrenergic receptor expressed in control cells. Enhancing TSHR internalization by β-arrestin 2 overexpression did not modify the reversibility of TSHR-cAMP signaling. These findings strengthen the view that the cointernalization of TSH-TSHR complexes to a signaling compartment present in thyroid, but not in HEK 293 cells, is responsible for persistent cAMP signaling.

Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells

Dissociation kinetics of 125I-nerve growth factor from cell surface receptors. Acceleration by unlabeled ligand and its relationship to negative cooperativity.

C G Thomas

Papers

Thyrotropin receptors in normal human thyroid. Nonclassical binding kinetics not explained by the negative cooperativity model.