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The higher order form merely improves the regression result to ~70%.
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Higher order forms have been defined with multiple time offset variables.
However, these higher order forms of ectodomain were unstable on mild reduction with DTT.
Hence, the data demonstrated that TSHR ectodomains were capable of forming higher order forms in the absence of the transmembrane domain.
These animals, however, have very limited cognitive functions in comparison to terrestrial mollusks, which are capable of higher order forms of learning and memory [8], [9].
The TSHR, in addition to undergoing intramolecular cleavage, also exists as dimers and higher order forms both in native tissue and transfected cells [13] [15].
We expressed untagged and tagged TSHR ectodomains (residues 1 412) in mammalian cells and found higher order forms of the receptor under non-reduced conditions.
Distinct bands of 250KD corresponding to higher order forms of the ectodomain was observed in untreated and TSH treated samples under non-reduced conditions (Figure 2A).
We had earlier established, by biochemical and biophysical approaches, that the full length TSHR is able to form dimeric and higher order forms in native and transfected cells [13], [16], [17].
Therefore, to understand the exact biological role of dimers and/or higher order forms of receptors it will be necessary to generate GPCRs that are expressed only as monomeric or multimeric receptor forms in model cell systems.
The blots when probed with a TSHR antibody M4 (RSR 4: recognizing residue 322 341) [27], revealed the presence of higher order forms in addition to the fully mature monomeric form of GPI: TSHR Ecd consisting of a doublet comprising of a 100KD form and less mature molecular species of 74KD as observed by others [28].
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