Functional analysis of a type 1 parathyroid hormone receptor intracellular tail mutant [KRK(484-6)AAA]: effects on second messenger generation and cellular targeting.
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.
Patterson
Eric K
EK
The Department of Biochemistry, University of Western Ontario, and The Lawson Health Research Institute, London, Ontario, Canada N6A 4V2.
Hodsman
Anthony B
AB
Hendy
Geoffrey N
GN
Canaff
Lucie
L
Bringhurst
F Richard
FR
Fraher
Laurence J
LJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D018374:LLC-PK1 Cells; D051379:Mice; D018613:Microscopy, Confocal; D009154:Mutation / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics* / Q000378:metabolism; D015290:Second Messenger Systems / Q000235:genetics*; D013552:Swine; D014162:Transfection
D019913:Nuclear Localization Signals; D044168:Receptor, Parathyroid Hormone, Type 1; D002118:Calcium