کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2781118 | 1153313 | 2010 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Functional analysis of a type 1 parathyroid hormone receptor intracellular tail mutant [KRK(484-6)AAA]: Effects on second messenger generation and cellular targeting
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شناسی تکاملی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The parathyroid hormone receptor type 1 (PTHR1) is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP) and primarily signals via intracellular pathways involving adenylyl cyclase and phospholipase C. The intracellular tail domain of the PTHR1 contributes to G protein subunit coupling that is important for second messenger signalling. In addition, the intracellular domain has a potential nuclear localization sequence (NLS) that, if functional, could point to an intracrine role for the receptor. In the present study, we have utilized 2 sets of constructs that employ either a [KRK(484-486)AAA](3Ala) mutation in the putative NLS or the non-mutant counterpart and included (a) the full-length rat PTHR1 with FLAG and c-myc epitope tags at the N-terminus and C-terminus, respectively (designated as PTHR1(3Ala)-TAG and PTHR1-TAG); and (b) only the putative NLS-containing intracellular domain (471-488), with green fluorescent protein (GFP) fused to the C-terminus (designated as GFP-(3Ala)471-488 or GFP-471-488). Porcine kidney LLC-PK1 cells stably expressing the PTHR1(3Ala)-TAG exhibited reduced signalling via both cAMP and cytosolic calcium transients in spite of greater cell surface expression relative to cells expressing PTHR1-TAG. We also examined the ability of the intracellular tail to influence the cellular localization of a heterologous protein. LLC-PK1 cells transiently transfected with GFP-471-488, exhibited increased fluorescence within the nucleus, relative to cells transfected with GFP alone that was not observed when cells were transiently transfected with the mutated construct, GFP-(3Ala)471-488. However, LLC-PK1 cells transiently transfected with either the full-length PTHR1-TAG or the PTHR1(3Ala)-TAG constructs did not exhibit nuclear localization of these receptors. Moreover, mouse osteoblast-like cells (MC3T3-E1) transiently expressing PTHR1-TAG also failed to demonstrate nuclear localization, although both full-length PTHR1 constructs exhibited plasma membrane immunofluorescence in both cell lines. Thus, the 484-486 sequence is critical for the full signalling responsiveness of the intact PTHR1, but the putative nuclear localization signal may not function as such within the intact receptor.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Bone - Volume 46, Issue 4, April 2010, Pages 1180-1187
Journal: Bone - Volume 46, Issue 4, April 2010, Pages 1180-1187
نویسندگان
Eric K. Patterson, Anthony B. Hodsman, Geoffrey N. Hendy, Lucie Canaff, F. Richard Bringhurst, Laurence J. Fraher,