Signaling Transduction

Signaling transduction

Answer 1)

The figure 1c explains the experiment that was chose to introduce this modification into the entire receptor sequence to demonstrate the importance of the 1-10 motif for CaM binding to the receptor and its signaling properties within living cells. In absence of agonist, 5-HT2C receptor did not coimmunoprecipitate with CaM in homogenates from HEK-293 cells cotransfected with Myc-5- HT2C receptor and GFP-CaM. In contrast, 5-HT2C receptor coimmunoprecipitated with GFP-CaM after acute exposure to 5-HT.

The negative control experiment conducted on the nontransformed HEK-293 cells revealed no enzymatic activity on estrone, showing that no 17HSD1 activity was initially present in the nontransformed cells. The positive control experiment showed that estrone was converted by the transformed cells, consistent with 17HSD1 activity. The fact that the pseudo-symmetry of C19 steroids allows alternative binding orientations is supported by the similar Km measured for the 3reduction and the 17oxidation of DHT. On the other hand, estrogens do not show the same level of symmetry as androgens, because they lack the C19-methyl group and possess a planar/aromatic A-ring. These features were found to be important in order to recognize and correctly orient estradiol in the active site.

The simultaneous 3 reduction and 17oxidation of DHT by 17HSD1 are additive in the inactivation of the most potent androgen. Therefore, it is possible that 17HSD1 can participate in reducing DHT levels in the peripheral intracrine tissues and act as a modulator of the occupancy of the androgen receptor in these tissues, especially in the human mammary gland where both 17-HSD1 and DHT are present. Future investigations on other enzymes able to distinguish between C18 and C19 steroids will reveal whether reverse binding orientation is the result of a more general discrimination mechanism or if it is specific to human 17HSD1. We speculate that alternative binding orientations may be a more general mechanism and could be found in other steroid-converting enzymes and receptors.

Answer 2)

MMP2 is a Zn+2 dependent endopeptidase, synthesized and secreted in zymogen form. The nascent form of the protein shows an N-terminal signal sequence ("pre" domain) that directs the protein to the endoplasmic reticulum. The pre domain is followed by a propeptide-"pro" domain that maintains enzyme-latency until cleaved or disrupted, and a catalytic domain that contains the conserved zinc-binding region. A hemopexin/vitronectin-like domain is also seen, that is connected to the catalytic domain by a hinge or linker region. The hemopexin domain is involved in TIMP (Tissue Inhibitors of Metallo-Proteinases) binding, the binding of certain substrates, membrane activation, and some proteolytic activities. It also shows a series of three head-to-tail cysteine-rich repeats within its catalytic domain. These inserts resemble the collagen-binding type II repeats of fibronectin and are required to bind and cleave collagen and elastin. The regulation of MMP-2 activity occurs at many levels, of which regulation through TIMP-2 and its cell surface receptor, MT1-MMP (MMP14) is critically decisive. At higher levels of TIMP-2, MT1-MMP forms a ternary complex with MMP-2 through, leaving no free MT1-MMP receptors, thereby inhibiting the activation ...